Parapoxvirus vectors

ABSTRACT

The present invention is in the field of viral immunotherapy. The invention provides new pseudocowpox (PCPV) viruses, in particular recombinant PCPV, composition thereof as well as their therapeutic use for preventing or treating diseases, and, notably, proliferative diseases like cancers and restenosis and infectious diseases such as chronic ones. The present invention also provides methods for generating and amplifying such a PCPV and a method for eliciting or stimulating and/or re-orienting an immune response using such a PCPV. More specifically, the invention provides an alternative to the existing poxvirus vectors such as MVA (Modified Virus Ankara) and may be largely used for the therapeutic vaccination.

TECHNICAL FIELD OF THE INVENTION

The present invention is in the field of viral immunotherapy. Theinvention provides new pseudocowpox (PCPV) viruses, in particularrecombinant PCPV, composition thereof as well as their therapeutic usefor preventing or treating diseases, and, notably, proliferativediseases like cancers and restenosis and infectious diseases. Thepresent invention also provides methods for generating and amplifyingsuch a PCPV and a method for eliciting or stimulating and/orre-orienting an immune response using such a PCPV. More specifically,the invention provides an alternative to the existing poxvirus vectorssuch as MVA (Modified Virus Ankara) and may be largely used for thetherapeutic vaccination.

BACKGROUND ART

Immunotherapy seeks to boost the host's immune system to help the bodyto eradicate pathogens and abnormal cells. Widely used in traditionalvaccination, immunotherapy is also being actively investigated as apotential modality for treating severe, chronic or life-threateningdiseases in an attempt to stimulate specific and innate immuneresponses. A vast number of immunotherapeutics have been described inthe literature for decades. In particular, several viral and non-viralvectors have now emerged, all of them having relative advantages andlimits making them more appropriate to certain indications (see forexample Cattaneo and Russell, 2017, PLOS Pathogensdoi:10.1371/journalppat.1006190; Kaufman et al., 2015, Nature ReviewsDrug Discovery 14: 642-661; Gomez et al., 2013 expert Rev Vaccines12(12): 1395-1416). A huge number of immunotherapy platforms are beingevaluated in clinical trials and the number of current clinical studiesbased on poxvirus therapy, whether oncolytic or not, reflects theirinteresting therapeutic potential. For example, recombinant vacciniavirus (VV)-based vectors are attractive candidates for their excellentsafety profile and their capacity to combine robust cellularantigen-specific immune responses with a generalized stimulation of theinnate immune system. TG4010 (or MVATG9931 with its research name) is atherapeutic cancer vaccine based on a modified vaccinia virus Ankara(MVA) coding for MUC1 tumor-associated antigen and human interleukin 2(IL-2). TG4010, in combination with first-line standard of carechemotherapy in advanced metastatic non-small-cell lung cancer (NSCLC),demonstrated efficacy in two different randomized and controlled phase2b clinical trials (Quoix et al., 2011, The Lancet Oncology 12(12):1125-33).

Parapoxviruses represent different candidates that can be used in vectorvaccines. Parapoxvirus belongs to the family Poxviridae and thesubfamily Chordopoxvirinae. Parapoxviruses are commonly known ascausative agents of dermal diseases in ruminants, leading to papularstomatitis and contagious pustular dermatitis, especially in the regionsof the lips, nostrils, oral mucosa, and teats. Like other members of thePoxviridae family, parapoxvirus are relatively large and envelopeddouble-stranded DNA viruses with ovoid geometries that can infectvertebrates including a wide selection of mammals and humans.Parapoxviruses have a unique spiral coat that distinguishes them fromother poxviruses.

As for poxviruses, viral replication of parapox is cytoplasmic. Entryinto the host cell is achieved by attachment of the viral proteins tohost glycosaminoglycans (GAGs) that mediates endocytosis of the virusinto the host cell. Fusion with the plasma membrane permits to releasethe core into the host cytoplasm. Early genes are transcribed in thecytoplasm by viral RNA polymerase. Early expression begins at 30 minutespost-infection. Intermediate phase triggers genomic DNA replication atapproximately 100 minutes post-infection. Late genes are then expressedfrom 140 min to 48 hours post-infection, producing all structuralproteins. Assembly of progeny virions starts in cytoplasmic viralfactories, producing a spherical immature particle. This virus particlematures into brick-shaped intracellular mature virion (IMV). IMV virioncan be released upon cell lysis or can acquire a second double membranefrom trans-Golgi and bud as external enveloped virion (EEV) hostreceptors, which mediates endocytosis. The virus exits the host cell byexisting in occlusion bodies after cell death and remains infectiousuntil it finds another host.

Replication-competent as well as inactivated Parapoxviruses are knownfor their immunomodulating properties (Schulze et al., 2009, VetMicrobiol. 137: 260-7). Parapoxvirus ovis (ORFV), the prototype speciesof the parapoxvirus genus, has been used successfully in veterinarymedicine for increasing general resistance in animal chronicallypersistent viral infections (see e.g. U.S. Pat. No. 6,365,393;WO97/32029 and US2003-0013076) as well as in human medicine for treatingHIV (WO2006/005529) and considered as oncolytic by some (Rintoul et al.,2012, Mol. Ther. 20(6): 1148-57). Various insertion sites wereidentified within the ORFV genome (WO97/37031). Notably, recombinantORFV encoding canine disempter virus (CDV) antigen were used as vaccineagainst CDV (WO2012/01145) and pseudorabies virus in pigs (Rooij et al.,2010, Vaccine 28(7): 1808-13). Zylexis®, formerly known as Baypamune®,which is a preparation of chemically inactivated ORFV derived fromstrain D1701 is used for the prophylaxis and therapeutic treatment ofinfectious diseases and for preventing stress-induced diseases inanimals. Inactivated ORFV was shown to induce plasmacytoid dendriticcells (pDC) probably through the engagement of a TLR-9 dependent pathway(Von Buttlar et al., 2014, PLOS One 9(8): e106188). More recently, Choiet al. (2017, Surgery, doi 10.1016/j.surg.2017.09.030) reported potentcytotoxic activities of a chimeric parapoxvirus in triple negativebreast cancer (TNBC) tumors. Active replication of the chimeric ORFvirus was detected in the tumor tissues 1 week after its injection andnatural killer (NK) cell infiltration was observed in the periphery ofvirus treated tumor tissues.

There is clearly an important need to develop effective approaches forthe treatment of life-threatening diseases such as cancers andinfectious diseases. Indeed, diseased cells have evolved potentimmunosuppressive mechanisms for eluding the immune system, posing amajor obstacle to effective immunotherapy. Hence, triggering both innateand specific immune mechanisms may be key to successful development ofmore effective immunotherapeutics.

The present invention relates to using Parapoxvirus as a vector for thedelivery of therapeutic genes. The inventors surprisingly discoveredthat Pseudocowpox virus (PCPV) offers remarkable advantages which makesit particularly appropriate for anti-cancer therapy considering itslimited pathogenicity and immune modulating properties. The inventorsdiscovered that PCPV provides a strong innate immune profile differentfrom those of other poxviruses as evidenced by its ability to activatesecretion of a number of cytokines and chemokines that stimulate immuneeffector cells at a level higher than conventional MVA and VV. Anothermember of Parapoxvirus genus, the bovine popular stomatitis virus(BPSV), showed similar effects in PBMCs as PCPV with a strong increaseof secreted IFN-alpha in supernatants. Moreover, increased CD86expression in human in vitro-derived M2 type macrophages suggests a roleof PCPV in reprogramming M2 macrophages towards a less suppressivephenotype. Finally, a recombinant PCPV virus engineered to expresstumor-associated antigens (TAA) was shown particularly effective tocontrol tumor growth and enhance survival in a syngenic animal model.The combination of PCPV with an anti-PD1 antibody showed statisticallysignificant improvement of tumor control in a “two tumor” model.

Due to the improved immunogenic properties exhibited by the PCPV andBPSV viruses, one may anticipate that parapoxvirus may be successfullyused as an alternative to other viral therapies for treating orpreventing proliferative diseases such as cancer and infectious diseases(e.g. chronic HBV). Thus, PCPV or BPSV is a candidate for a therapeuticvaccine which could have effects on the tumor environment.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a pseudocowpoxvirus (PCPV)wherein said PCPV comprises at least one foreign nucleic acid insertedin its genome.

In one embodiment, said PCPV is obtained from the wild-type TJS strainas identified by ATCC reference number ATCC VR-634™ or from a virusstrain of the same or similar name or functional fragments and variantsthereof.

In another embodiment, the PCPV may be further defective for a viralfunction encoded by the PCPV genome and preferably for a non-essentialviral function and, more preferably for a viral gene function encoded atthe insertion site of said foreign nucleic acid.

In a further embodiment, said foreign nucleic acid encodes a polypeptideselected from the group consisting of polypeptides that compensate fordefective or deficient proteins in a subject, polypeptides that actthrough toxic effects to limit or remove diseased cells from the bodysuch as suicide gene products; polypeptides capable of potentiatinganti-tumor efficacy such as armed gene products; and polypeptidescapable of inducing or activating an immune response such asimmunostimulatory and antigenic polypeptides. Preferably, theimmunostimulatory polypeptide is selected from the group consisting ofcytokines, such as interleukins, chemokines, interferons, tumor necrosisfactor, colony-stimulating factors, APC-exposed proteins, polypeptideshaving an anti-angiogenic effect and polypeptides that affect theregulation of cell surface receptors such as agonists or antagonists ofimmune checkpoints with a specific preference for an interleukin or acolony-stimulating factor and, in particular GM-CSF or an agonistOX40-directed antibody. Also preferred is a cancer antigen such as theMUC-1 antigen, a viral antigenic polypeptide such as the HPV-16 E7antigens or HBV antigens.

In still a further embodiment, the at least one foreign nucleic acid isoperably linked to suitable regulatory elements for expression in adesired host cell or subject.

In an additional embodiment, the at least one foreign nucleic acid isinserted in the VEGF locus.

In another aspect, the present invention further provides a method forgenerating the PCPV of the invention, by homologous recombinationbetween a transfer plasmid comprising the foreign nucleic acid flankedin 5′ and 3′ with PCPV sequences respectively present upstream anddownstream the insertion site and a PCPV genome, wherein said methodcomprises a step of generating said transfer plasmid and a step ofintroducing said transfer plasmid into a suitable host cell, notablytogether with a PCPV virus comprising the flanking sequence present inthe transfer plasmid.

In one embodiment, the site of insertion of the at least one foreignnucleic acid in the PCPV genome is in a viral gene, with a preferencefor a non-essential viral gene, in an intergenic region, in a portion ofthe PCPV genome which does not encode gene products or in a duplicatedlocus and, preferably, the VEGF locus.

In one embodiment, the transfer plasmid further comprises one or moreselection and/or detectable gene to facilitate identification of therecombinant PCPV with a preference for the selection GPT gene and/or adetectable gene encoding GFP, e-GFP or mCherry.

In a further aspect, the present invention further relates to a methodfor amplifying the PCPV of the invention or generated by the method ofthe invention, comprising the steps of a) preparing a producer cellline, b) transfecting or infecting the prepared producer cell line, c)culturing the transfected or infected producer cell line under suitableconditions so as to allow the production of the virus, d) recovering theproduced virus from the culture of said producer cell line andoptionally e) purifying said recovered virus.

In still a further aspect, the present invention relates to acomposition comprising a therapeutically effective amount of the PCPV ofthe invention or amplified by the method according to the invention anda pharmaceutically acceptable vehicle. Preferably, the composition isformulated in individual doses comprising from approximately 10³ toapproximately 10¹² pfu. Preferably, it is formulated for intravenous,intramuscular, subcutaneous or intratumoral administration.

In one embodiment, the composition is for use for treating or preventingdiseases or pathological condition caused by a pathogenic organism or anunwanted cell division.

In still a further aspect, the invention provides a method of treatmentand a method for inhibiting tumor cell growth comprising administeringthe composition of the invention to a subject in need thereof in anamount sufficient to treat or prevent a disease or a pathologicalcondition caused by a pathogenic organism or an unwanted cell division.

In one embodiment, said cancer is selected from the group consisting ofrenal cancer, prostate cancer, breast cancer, colorectal cancer, lungcancer, liver cancer, gastric cancer, bile duct carcinoma, endometrialcancer, pancreatic cancer and ovarian cancer and a cancer thatoverexpresses MUC1.

In another embodiment, said method or use is carried out in conjunctionwith one or more other therapeutic agents selected from the groupconsisting of surgery, radiotherapy, chemotherapy, cryotherapy, hormonaltherapy, toxin therapy, immunotherapy, cytokine therapy, targeted cancertherapy, gene therapy, photodynamic therapy and transplantation. In afurther embodiment, said method or use is carried out according to aprime boost approach which comprises sequential administrations of apriming composition(s) and a boosting composition(s).

In a further aspect, the invention relates to a method for eliciting orstimulating and/or re-orienting an immune response comprisingadministering the composition of the invention to a subject in needthereof, in an amount sufficient to activate the subject's immunity.

In one embodiment, the method results in at least one the followingproperties (a) the secretion of high levels of IFN-alpha from PBMC; (b)the activation of monocyte-derived dendritic cells; (c) the induction ofT cell proliferation (e.g. as indirectly reflected by highly granzyme B+T cells); (d) a better cytokine/chemokine profile in MDSC; (e)activation of APC; (f) a M2 to M1 conversion of human macrophages;and/or (g) the induction of immunity through a TLR9-mediated pathway orothers innate immunity-stimulating pathways.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates IFN-alpha secretion in PBMCs from 2 donors in twodifferent experiments (Exp 1 and exp 2) at different MOI (MOI 0.1; MOI 1and MOI 5). Mock represents the negative control whereas the differentviruses tested are Parapoxviruses (pseudocowpoxvirus (PCPV) andParapoxvirus ovis (ORFV)), MVA (an empty MVA vector named MVAN33.1),cowpox virus (CPX), Copenhagen vaccinia virus (Copwt), fowlpox (FPV),Myxomavirus (MYXV), Swine pox (SWPV), raccoonpoxvirus (RCN), Cotiavirus(CTV) and Yaba-like disease virus (YLDV).

FIG. 2 illustrates expression levels of the activation marker CD86measured by flow cytometry in virus-infected moDCs obtained from threedifferent donors. Shown is median fluorescence intensity (MedFl) forCD86 in moDCs (3 donors), observed 16 h after infection with the emptyMVA vector MVAN33.1 or PCPV at MOI 0.03, 0.3 or 1 or mock as negativecontrol. Expression levels were measured by staining with anti-CD86-PEquantification of the cell-bound signal by flow cytometry.

FIG. 3 illustrates the cytokine levels measured by Luminex analysis inthe supernatant of CD163+ CD206+ M2 macrophages generated from PBMCs asdescribed herein following incubation with MVAN33.1 or PCPV at MOI 5, 1and 0.3.

FIG. 4 illustrates A) Relative CTL proliferation alone or in co-culturewith MDSCs derived in the presence of 20 ng/ml GM-CSF, 10 ng/ml IL4 and1 μM prostaglandin E2 (MDSC/T ratio of ½) after treatment with PCPV atMOI 0.03, 0.3 or 3. B) Relative Granzyme B expression in highlyproliferating T cells alone or in co-culture with MDSC (co-cultured asindicated in A) treated with PCPV at indicated MOI. C) IFN-alphasecretion after treatment of MDSCs with PCPV overnight at indicated MOI.

FIG. 5 illustrates A) Relative CTL proliferation alone or in co-culturewith MDSCs derived in the presence of 10 ng/ml GM-CSF and 10 ng/ml IL-6(MDSC/T ratio of ½ and 1/1) after treatment with PCPV at MOI 0.03, 0.3or 3. B) Relative Granzyme B expression in proliferating T cells aloneor in co-culture with MDSC (co-cultured as indicated in A) treated withPCPV at indicated MOI.

FIG. 6 illustrate the evolution of tumor growth and animal survival inMC38 tumor model. The animals were divided in three groups of ten mice.For all groups, syngeneic C57BL/6 mice were injected subcutaneously with2·10⁶ MC38 cells according to the protocol shown in (A). The injectionsite was labeled with a permanent marker. The animal received threeintratumoral injections of 1·10⁷ pfu of MVA-HPV16E7m (B) orPCPV-HPV16E7m (C) or buffer (D), respectively 2 days after cellimplantation (at the cell line injection site) and in the emerging tumorat day 9 and 16. Tumor growth was estimated by measuring tumor volumesover time. (E): Overall survival (OS) rates represented as Kaplan-Meiercurves. Solid line (-) represents animal group treated with MVA-HPV16E7m(MVATG14197), dashed line (- -) the group treated with PCPV-HPV16E7m(PCPV19178) and dotted line (. .) the one treated with buffer (S08).

FIG. 7 illustrates the PCPV effect on viability of MC38 cells. 2×10⁴cells were plated and infected at MOI 1, 10⁻¹, 10⁻² and 10⁻³ with eitherPCPV-GFP; VV-GFP (designated VVTG) as positive control and mock asnegative control. Five days later, cells were harvested by scraping andnumber and proportion of living cells were determined using TrypanBlue-staining and the cell counter Vicell.

FIG. 8 illustrates the local cytokine and chemokine profile after scinjection of PCPV or MVA or buffer (as negative controls). 5×10⁵ pfu ofvirus/flank were injected sc in both shaved flanks of four mice pergroup. For each mouse, skin from both flanks was taken after 16 hours,and mechanically dissociated preserving the cells (liberation ofinterstitial/extracellular analytes). After centrifugation at 300 g,supernatants were transferred in Eppendorf tubes and centrifuged at18000 g in the cold. Cleared supernatant was analyzed with Procartaplexmouse chemokine and cytokine multiplex kits using a MagPix deviceaccording to the manufacturer's recommendations.

FIG. 9 illustrates the effect of PCPV on TIL composition. The percentageof neutrophils was detected in the population of CD11 b⁺ Ly6G⁺ CD45⁺cells (A). TAMs were identified as CD11c− subpopulation within Ly6G⁻CD11b⁺ cells. Various subpopulations were stratified with MHC II and Ly6C.“TAM C” according to Brauner et al, were characterized as MHC II^(lo)and Ly6C⁺.

FIG. 10 illustrates (A) the generation of HPV16 E7-specific T cells inpooled spleens (ELISPOT) after stimulation with R9F peptide, (B) thegeneration of MVA-specific T cells after stimulation with T8V, (C) theappearance of the CD3^(dim)CD8^(dim) T cell population in pooled lungsof mice repeatedly treated intravenously (iv) with the indicated virusand (D) the generation of HPV-16 E7-specific T cells in pooled lungswithin the CD3^(dim)CD8^(dim) T cell population.

FIG. 11 illustrates the fold induction (specific peptide versus controlpeptide) of the appearance of HPV16 E7-specific T cells within theCD3^(dim)CD8^(dim) T cell population measured by ICS upon infection withan empty MVA (MVATGN33.1), HPV16E7-encoding MVA (MVATG14197) andHPV16E7-encoding PCPV.

FIG. 12 illustrates a tumor control experiment in MC38-bearing mice uponintratumoral injection of 1×10⁷ pfu of PCPV (A) or buffer (E) andintraperitoneal injection of depleting antibodies anti-Ly6G (B),anti-CD8 (C) and anti-CD4 (D) to deplete mouse neutrophils, CD8+ cellsand CD4+ cells, respectively. Each group comprise 13 mice.

FIG. 13 illustrates ELISPOT analysis on splenocytes from survivor micehaving received PCPV injections and showing reduced or resolved MC38tumors at day 34. Spleens from 5 survivor animals (PCPV survivor pool)and of two individual survivors (PCPV survivor) and of naïve mice(Naives) were collected to isolate lymphocytes from the splenocytesuspensions. One10⁷ cells per mL were plated in ELISA plates andstimulated with Mitomycin C-treated MC38 cells or irrelevant I8L peptideor with complete medium (medium). Spots were counted with an ELISPOTreader (CTL Immunoqpot reader). Each condition was tested in quadriplateand the results expressed as the mean number of spot-forming units (sfu)per 1×10⁶ splenic lymphocytes.

FIG. 14 illustrates a tumor control experiment in TC1-bearing C57BL/6mice according to a prime/boost setting. TC1 cells were subcutaneouslyimplanted in the flank of C57BL/6 mice. After 14 days, tumor-bearingmice were randomized (10 mice per group), and intratumorally injectedwith 1·10⁶ pfu of MVA-HPV16E7 (A and B) or PCPV-HPV16E7 (C) or buffer(D) (priming injection). One week later, mice were boosted withintravenous injection of 1×10⁶ pfu of the counterpart virus, i.e.PCPV-HPV16E7 (D) or MVA-HPV16E7 (A and C) or buffer (B in the bufferprimed mice). Tumor growth were followed overtime.

FIG. 15 illustrates a tumor control experiment in MC38-bearing mice uponintratumoral injection of 1×10⁷ pfu of PCPV (A) or VV (B) either asstand-alone (dark grey) or in combination with the murine anti-PD1(intraperitoneal injection) (medium grey). Control group received S08buffer (lightest grey) or anti-PD1 antibody alone (light grey). Eachgroup comprises 13 mice.

FIG. 16 illustrates IFN-alpha secretion in PBMCs obtained from arepresentative donor and infected with MVA, PCPV or BPSV at MOI 0.3.Mock represents the negative control and ODN2216 a CpG type TLR9 ligandas a control of immunostimulatory effect.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,unless the context clearly dictates otherwise. For example, the term “acell” includes a plurality of cells, including mixtures thereof.

The term “one or more” refers to either one or a number above one (e.g.2, 3, 4, etc.).

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange.

As used herein, when used to define products and compositions, the terms“comprising” (and any form of comprising, such as “comprise” and“comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are open-ended and do not exclude additional,unrecited elements or method steps. The expression “consistingessentially of” means excluding other components or steps of anyessential significance. Thus, a composition consisting essentially ofthe recited components would not exclude traces, contaminants andpharmaceutically acceptable carriers. “Consisting of” shall meanexcluding more than trace elements of other components or steps.

The terms “polypeptide”, “peptide” and “protein” refer to polymers ofamino acid residues which comprise at least nine or more amino acidsbonded via peptide bonds. The polymer can be linear, branched or cyclicand may comprise naturally occurring and/or amino acid analogues and itmay be interrupted by non-amino acids. As a general indication, if theamino acid polymer is more than 50 amino acid residues, it is preferablyreferred to as a polypeptide or a protein whereas if it is 50 aminoacids long or less, it is referred to as a “peptide”.

Within the context of the present invention, the terms “nucleic acid”,“nucleic acid molecule”, “polynucleotide” and “nucleotide sequence” areused interchangeably and define a polymer of any length of eitherpolydeoxyribonucleotides (DNA) (e.g. cDNA, genomic DNA, plasmids,vectors, viral genomes, isolated DNA, probes, primers and any mixturethereof) or polyribonucleotides (RNA) (e.g. mRNA, antisense RNA, SiRNA)or mixed polyribo-polydeoxyribonucleotides. They encompass single ordouble-stranded, linear or circular, natural or synthetic, modified orunmodified polynucleotides. Moreover, a polynucleotide may comprisenon-naturally occurring nucleotides and may be interrupted bynon-nucleotide components.

The terms “variant” “analog” “derivative” and the like can be usedinterchangeably to refer to a component (polypeptide, nucleic acid,virus, etc) exhibiting one or more modification(s) with respect to thenative counterpart. Any modification(s) can be envisaged, includingsubstitution, insertion and/or deletion of one or more nucleotide/aminoacid residue(s). Preferred are variants that retain a degree of sequenceidentity of at least 75%, advantageously at least 80%, desirably atleast 85%, preferably at least 90%, more preferably at least 95%, andeven more preferably at least 98% identity after optimal globalalignment with the sequence of the native counterpart, i.e. afteralignment of the sequences to be compared taken in their entirety overtheir entire length. For illustrative purposes, “at least 75% identity”means 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In ageneral manner, the term “identity” refers to an amino acid to aminoacid or nucleotide to nucleotide correspondence between two polypeptidesor nucleic acid sequences. The percentage of identity between twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps which need to beintroduced for optimal alignment and the length of each gap. Variouscomputer programs and mathematical algorithms are available in the artto determine the percentage of identity between amino acid sequences,such as for example the Blast program available at NCBI or ALIGN inAtlas of Protein Sequence and Structure (Dayhoffed, 1981, Suppl., 3:482-9). Programs for determining identity between nucleotide sequencesare also available in specialized data base (e.g. Genbank, the WisconsinSequence Analysis Package, BESTFIT, FASTA and GAP programs). For optimalglobal alignments, the algorithm of Needleman and Wunsch (Needleman andWunsch. J. Mol. Biol. 48,443-453, 1970) may be used, for instance usingthe Emboss Needle software available athttps://www.ebi.ac.uk/Tools/psa/emboss_needle/. This software reads twoinput sequences and writes their optimal global sequence alignment tofile. It uses the Needleman-Wunsch alignment algorithm to find theoptimum alignment (including gaps) of two sequences along their entirelength. The algorithm uses a dynamic programming method to ensure thealignment is optimum, by exploring all possible alignments and choosingthe best. A scoring matrix is read that contains values for everypossible residue or nucleotide match. Needle finds the alignment withthe maximum possible score where the score of an alignment is equal tothe sum of the matches taken from the scoring matrix, minus penaltiesarising from opening and extending gaps in the aligned sequences. Thesubstitution matrix and gap opening and extension penalties areuser-specified. In the context of the invention, in order to obtain anoptimal global alignment, the Emboss Needle software may be used withdefault parameters, i.e.:

-   -   For amino acid sequences: “Gap open”=10.0, “Gap extend”=0.5,        “End gap penalty”=“false”, “End gap open”=10.0, “End gap        extend”=0.5, and a “Blosum 62” matrix;    -   For nucleotide sequences: “Gap open”=10.0, “Gap extend”=0.5,        “End gap penalty”=“false”, “End gap open”=10.0, “End gap        extend”=0.5, and a “DNAfull” matrix.

As used herein, the term “host cell” should be understood broadlywithout any limitation concerning particular organization in tissue,organ, or isolated cells. Such cells may be of a unique type of cells ora group of different types of cells such as cultured cell lines, primarycells and dividing cells. In the context of the invention, the term“host cells” refers more particularly to eukaryotic cells and, notably,to mammalian (e.g. human or non-human) cells as well as to cells capableof producing the PCPV virus of the invention (e.g. producer cell). Thisterm also includes cells which can be or has been the recipient of thevirus described herein as well as progeny of such cells.

The terms “virus”, ‘viral particle”, “viral vector” and virion” are usedinterchangeably and are to be understood broadly as meaning a vehiclecomprising at least one element of a wild-type virus genome that may bepackaged into a viral particle (also designated as a viral vector) or tothe viral particle itself. Usually, a virus comprises a DNA or RNA viralgenome packaged into a viral capsid and, in the case of an envelopedvirus, lipids and other components (e.g. host cell membranes, etc). Thepresent invention encompasses wild-type and engineered viruses. Asmentioned just above, the term “virus” and the like has to be understoodbroadly as including viral vector (e.g. DNA viral vector) as well asviral particles generated thereof. The term “infectious” refers to theability of a virus to infect and enter into a host cell or subject.

The term “naturally occurring” “native” or “wild type” is used todescribe a biological molecule or organism that can be found in natureas distinct from being artificially produced by man. For example, anaturally occurring, native or wild-type virus refers to a virus whichcan be isolated from a source in nature or obtained from specificcollections (e.g. ECCAC, ATCC, CNCM, etc). A biological molecule or anorganism which has been intentionally modified by man in the laboratoryis not naturally occurring. Representative examples of non-naturallyoccurring viruses include, among many others, recombinant virusesengineered by insertion of one or more nucleic acid(s) of interest inthe viral genome and/or defective virus resulting from one or moremodification(s) in the viral genome (e.g. total or partial deletion of aviral gene).

The term “obtained from”, “originating” or “originate” is used toidentify the original source of a component (e.g. a polypeptide, nucleicacid molecule, virus, etc) but is not meant to limit the method by whichthe component is made which can be, for example, by chemical synthesis,homologous recombination, recombinant means or any other means.

The term “treatment” (and any form of treatment such as “treating”,“treat”) as used herein encompasses prophylaxis (e.g. preventive measurein a subject at risk of having the pathological condition) and/ortherapy (e.g. in a subject diagnosed as having the pathologicalcondition), optionally in association with conventional therapeuticmodalities. The result of the treatment is to slow down, cure,ameliorate or control the progression of the targeted pathologicalcondition. For example, a subject is successfully treated for a cancerif after administration of a PCPV as described herein, alone or incombination, the subject shows an observable improvement of its clinicalstatus.

The term “administering” (or any form of administration, such as“administered”) as used herein refers to the delivery to a subject of atherapeutic agent such as the PCPV described herein.

The term “subject” generally refers to an organism for whom any productand method of the invention is needed or may be beneficial. Typically,the subject is a mammal, particularly a mammal selected from the groupconsisting of domestic animals, farm animals, sport animals, andprimates. Preferably, the subject is a human who has been diagnosed ashaving or at risk of having a pathological disease (e.g. a cancer). Theterms “subject” and “patients” may be used interchangeably whenreferring to a human organism and encompasses male and female. Thesubject to be treated may be a new-born, an infant, a young adult, anadult or an elderly.

Pseudocowpox Virus

In a first aspect, the present invention provides a pseudocowpoxvirus(PCPV) comprising at least one foreign nucleic acid inserted in itsgenome. In one embodiment said PCPV is for use for treating disease suchas proliferative or infectious diseases as described hereinafter.

The term “pseudocowpox virus” or “PCPV” is used herein according to itsplain ordinary meaning within Virology and refers to a member of thePoxviridae family which replicates in the cytoplasm of its host andbelonging to the Parapoxvirus genus. PCPV possesses a linear anddouble-stranded DNA genome, typically of 130-150 kilobases. The presentinvention encompasses naturally occurring forms of pseudocowpox virus ofany strain as well as variants thereof which may be modified for variouspurposes including those described herein.

The parapoxvirus genus encompasses a series of different speciesincluding Parapoxvirus ovis (ORFV), pseudocowpox virus (PCPV) and bovinepapular stomatitis virus (BPSV) and in each species different strainshave been described in the art with disclosure of complete or partialgenomic sequences; e.g. 01701, NZ2, NZ7, IA82, F07.821R, F09.1160S andSA00 strains of ORFV and AR02 and V660 strains of bovine papularstomatitis virus. Representative examples of suitable PCPV strains foruse herein include, without limitation, YG2828 (Genbank accession numberLC230119), F07.801R (Genbank accession number JF773693), F10.3081C(Genbank accession number JF773695), F07.798R (Genbank accession numberJF773692), F99.177C (Genbank accession number AY453678), IT1303/05(Genbank accession number JF800906), F00.120R (Genbank accession numberGQ329669; Tikkanen et al., 2004, J. Gen. Virol. 85: 1413-8) and TJS(also called VR634; Genbank accession number GQ329670; Friedman-Kien etal., 1963, Science 140: 1335-6; available at ATCC under accession numberVR634). Such strains may have morphological, structural and/or geneticdifferences each other, e.g., in terms of ITR length, number ofpredicted genes and/or G C rich content (see e.g. Hautaniemi et al.,2010, J. Gen. Virol. 91: 1560-76).

In the context of the present invention, preference is given to the PCPVspecies. In a preferred embodiment, the PCPV virus of the presentinvention is obtained from the wild-type TJS strain as identified byATCC reference number ATCC VR-634™ or from a virus strain of the same orsimilar name and functional fragments and variants thereof. Preferably,such a variant maintains at least 75% identity at the nucleotide oramino acid level with at least a segment of 10 kilobase (e.g. acontinuous sequence of 10 kb) in the wild-type TJS pseudocowpox virusgenome.

Exemplary modifications that are appropriate in the context of thepresent invention include without any limitation insertion(s),substitution(s) and/or deletion(s) of one or more nucleotide(s) withinthe PCPV genome with the goal of modulating (e.g. increase or decrease)expression of one or more viral gene or virus infectivity when comparedto the wild-type pseudocowpox virus (e.g. TJS strain) or increasing itstherapeutic efficacy (e.g. increase the ability of the PCPV virus todifferentially express in diseased cells relative to healthy cells) orinsertion of one or more foreign nucleic acid(s) of therapeutic interestor generating a chimeric virus containing PCPV genomic fragment(s) withones obtained from a different virus origin.

In one and preferred embodiment, the PCPV of the invention comprisesinserted in its genome at least one foreign nucleic acid (e.g. resultingin a recombinant PCPV virus). Alternatively, or in combination, the PCPVvirus may be defective for a viral function encoded by the PCPV genome(e.g. a non-essential viral function), preferably for a viral genefunction encoded at the insertion site(s) of the foreign nucleic acid.

Recombinant PCPV

The term “recombinant” as used herein in connection with the virus ofthe present invention indicates that the virus has been modified by theintroduction of at least one foreign nucleic acid (also calledrecombinant gene or nucleic acid), notably a nucleic acid of therapeuticinterest as described herein. In the context of the invention, the“foreign nucleic acid” that is inserted in the PCPV genome is not foundin or expressed by a naturally-occurring PCPV genome. Nevertheless, theforeign nucleic acid can be homologous or heterologous to the subjectinto which the recombinant PCPV is introduced. More specifically, it canbe of human origin or not (e.g. of bacterial, yeast or viral originexcept PCPV). Advantageously, said foreign nucleic acid encodes apolypeptide or is a nucleic acid sequence capable of binding at leastpartially (by hybridization) to a complementary cellular nucleic acid(e.g., DNA, RNA, miRNA) present in a diseased cell with the aim ofinhibiting a gene involved in said disease. A polypeptide is understoodto be any translational product of a polynucleotide regardless of size,and whether glycosylated or not, and includes peptides and proteins.Such a foreign nucleic acid may be a native gene or portion(s) thereof(e.g. cDNA), or any variant thereof obtained by mutation, deletion,substitution and/or addition of one or more nucleotides.

In a preferred embodiment, the foreign nucleic acid encodes apolypeptide which is capable of providing a therapeutic or prophylacticactivity when administered appropriately to a subject (i.e. apolypeptide of therapeutic interest), leading to a beneficial effect onthe course or a symptom of the pathological condition to be treated. Avast number of polypeptides of therapeutic interest may be envisaged. Inone embodiment, the foreign nucleic acid encodes a polypeptide selectedfrom the group consisting of polypeptides that compensate for defectiveor deficient proteins in a subject, polypeptides that act through toxiceffects to limit or remove diseased cells from the body (e.g. suicidegene products); polypeptides capable of potentiating anti-tumor efficacy(e.g. armed gene products); and polypeptides capable of inducing oractivating an immune response (such as immunostimulatory and antigenicpolypeptides). A foreign nucleic acid encoding a detectable gene productmay also be useful in the context of the invention.

Immunostimulatory Polypeptides

As used herein, the term “immunostimulatory polypeptide” refers to apolypeptide which has the ability to stimulate the immune system, in aspecific or non-specific way. A vast number of polypeptides are known inthe art for their ability to exert an immunostimulatory effect. Examplesof suitable immunostimulatory proteins in the context of the inventioninclude without limitation cytokines, with a specific preference forinterleukins (e.g. IL-2, IL-6, IL-12, IL-15, IL-24), chemokines (e.g.CXCL10, CXCL9, CXCL11), interferons (e.g. IFNα, IFNβ, IFNγ), tumornecrosis factor (TNF), colony-stimulating factors (e.g. GM-CSF, C-CSF,M-CSF . . . ), APC (for Antigen Presenting Cell)-exposed proteins (e.g.B7.1, B7.2 and the like), polypeptides having an anti-angiogenic effect(e.g. inhibitors of Vascular Endothelial Growth Factor such asbevacizumab or ranibizumab) and polypeptides that affect the regulationof cell surface receptors such as agonists or antagonists of immunecheckpoints (e.g. antibodies directed to PD1 or its ligand (e.g.anti-PD-L1), CTLA4, LAG3, OX40 etc). In a preferred embodiment, theimmunostimulatory polypeptide is an interleukin or a colony-stimulatingfactor (e.g. GM-CSF) or an agonist OX40-directed antibody.

Antigenic Polypeptides

As illustrated in the Example section, PCPV can stimulate innate(non-specific) immunity of the injected subject. Therefore, combiningthis effect with expression of an antigenic polypeptide may provide avirus capable of stimulating both innate and specific immune responses.This can be of importance for obtaining an effective anti-cancerresponse and for vaccination purposes for providing protection against aspecific pathogen. Such effect may last for weeks.

The term “antigenic” refers to the ability to induce or stimulate ameasurable immune response in a subject into which the recombinant PCPVencoding the polypeptide qualified as antigenic has been introduced. Thestimulated or induced immune response against the antigenic polypeptideexpressed by said recombinant PCPV can be humoral and/or cellular (e.g.production of antibodies, cytokines and/or chemokines involved in theactivation of effector immune cells). The stimulated or induced immuneresponse usually contributes in a protective effect in the administeredsubject. A vast variety of direct or indirect biological assays areavailable in the art to evaluate the antigenic nature of a polypeptideeither in vivo (animal or human subjects), or in vitro (e.g. in abiological sample). For example, the ability of a particular antigen tostimulate innate immunity can be performed by for example measurement ofthe NK/NKT-cells (e.g. representativity and level of activation), aswell as, IFN-related cytokine and/or chemokine producing cascades,activation of TLRs and other markers of innate immunity (Scott-Algara etal., 2010 PLOS One 5(1), e8761; Zhou et al., 2006, Blood 107, 2461-2469;Chan, 2008, Eur. J. Immunol. 38, 2964-2968). The ability of a particularantigen to stimulate a cell-mediated immune response can be performedfor example by quantification of cytokine(s) produced by activated Tcells including those derived from CD4+ and CD8+ T-cells using routinebioassays (e.g. characterization and/or quantification of T cells byELISpot, by multiparameters flow cytometry, ICS, by cytokine profileanalysis using multiplex technologies or ELISA), by determination of theproliferative capacity of T cells (e.g. T cell proliferation assays by[³H] thymidine incorporation assay), by assaying cytotoxic capacity forantigen-specific T lymphocytes in a sensitized subject or by identifyinglymphocyte subpopulations by flow cytometry and by immunization ofappropriate animal models, as described herein.

It is contemplated that the term antigenic polypeptide encompassesnative antigen as well as fragment (e.g. epitopes, immunogenic domains,etc) and variant thereof, provided that such fragment or variant iscapable of being the target of an immune response. Preferred antigenicpolypeptides for use herein are tumor-associated antigens and antigensof pathogenic organisms (bacteria, viruses, parasites, fungi, viroids orprions). It is within the scope of the skilled artisan to select the oneor more antigenic polypeptide that is appropriate for treating aparticular pathological condition.

In one embodiment, the antigenic polypeptide(s) encoded by the PCPVvirus is/are cancer antigen(s) (also called tumor-associated antigens)that is associated with and/or serve as markers for cancers. Cancerantigens encompass various categories of polypeptides, e.g. those whichare normally silent (i.e. not expressed) in healthy cells, those thatare expressed only at low levels or at certain stages of differentiationand those that are temporally expressed such as embryonic and foetalantigens as well as those resulting from mutation of cellular genes,such as oncogenes (e.g. activated ras oncogene), proto-oncogenes (e.g.ErbB family), or proteins resulting from chromosomal translocations.

Some non-limiting examples of cancer antigens include, withoutlimitation, MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV),adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectalassociated antigen (CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA)and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, ProstateSpecific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, andPSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family (e.g. MUC1, MUC16, etc; seee.g. U.S. Pat. No. 6,054,438; WO98/04727; or WO98/37095), HER2/neu,p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin,beta-catenin and gamma-catenin, p120ctn, gp100.sup.Pmel117, PRAME,NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin,Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, Smadfamily of cancer antigens brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.

The cancer antigens may also encompass antigens encoded by pathogenicorganisms that are capable of inducing a malignant condition in asubject (especially chronically infected subject) such as RNA and DNAtumor viruses (e.g. HPV, HCV, HBV, EBV, etc) and bacteria (e.g.Helicobacter pilori).

Viral antigenic polypeptides for use in this invention include forexample antigens from hepatitis virus A, B, C, D or E, immunodeficiencyvirus (e.g. HIV), herpes viruses (HSV), cytomegalovirus, varicellazoster, papilloma virus (HPV), Epstein Barr virus (EBV), influenzavirus, para-influenza virus, adenovirus, coxsakie virus, picorna virus,rotavirus, respiratory syncytial virus, poxvirus, rhinovirus, rubellavirus, papovirus, mumps virus, measles virus. Some non-limiting examplesof HIV antigens include gp120 gp40, gp160, p24, gag, pol, env, vif, vpr,vpu, tat, rev, nef tat, nef. Some non-limiting examples of human herpesvirus antigens include gH, gL gM gB gC gK gE or gD or Immediate Earlyprotein such asICP27, ICP47, ICP4, ICP36 from HSV1 or HSV2. Somenon-limiting examples of cytomegalovirus antigens include gB. Somenon-limiting examples of derived from Epstein Barr virus (EBV) includegp350 and the EBV-encoded nuclear antigen (EBNA)-1. Some non-limitingexamples of Varicella Zoster Virus antigens include gp1, 11, 111 and1E63. Some non-limiting examples of hepatitis C virus (HCV) antigensincludes E1 or E2 env protein, core protein, NS2, NS3, NS4a, NS4b, NS5aand NS5b. Some non-limiting examples of hepatitis B virus (HBV) antigensincludes polymerase, core and env polypeptide (e.g., the combination ofHBV antigens described in WO2013/007772). Some non-limiting examples ofhuman papilloma virus (HPV) antigens include L1, L2, E1, E2, E3, E4, E5,E6, E7. Antigens derived from other viral pathogens, such as RespiratorySyncytial virus (e.g. F and G proteins), parainfluenza virus, measlesvirus, mumps virus, flaviviruses (e.g. Yellow Fever Virus, Dengue Virus,Tick-borne encephalitis virus, Japanese Encephalitis Virus) andInfluenza virus cells (e.g. HA, NP, NA, or M proteins) can also be usedin accordance with the present invention. In a preferred embodiment, thePCPV of the invention is engineered to encode and express HPV-16 orHPV-18 E6 and/or E7 antigens.

Bacterial antigenic polypeptides include for example antigens fromMycobacteria causing TB and leprosy, pneumocci, aerobic gram negativebacilli, mycoplasma, staphyloccocus, streptococcus, salmonellae,chlamydiae, neisseriae and the like.

Parasitic antigenic polypeptides include for example antigens frommalaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasisand filariasis.

Preferred antigenic polypeptides for expression by the PCPV of thepresent invention are

-   -   the MUC-1 antigen aberrantly glycosylated and overexpressed in a        variety of epithelial cancers;    -   the HPV-16 E7 antigen, in particular a non-oncogenic variant        thereof.    -   HBV antigens, in particular a fusion comprising HBV polymerase,        HBV core protein and HBsAg immunogenic domains (e.g. as        described in WO2013/007772)

Suicide Gene Products

The term “suicide gene product” refers to a polypeptide able to converta precursor of a drug, also named “prodrug”, into a cytotoxic compound.Examples of suicide gene products and corresponding prodrugs aredisclosed in the following table:

TABLE 1 Suicide gene product Prodrug Thymidine Kinase Ganciclovir;Ganciclovir elaidic acid ester; penciclovir; Acyclovir; Valacyclovir;(E)-5-(2- bromovinyl)-2′-deoxyuridine; zidovudine; 2′-Exo-methanocarbathymidine Cytosine deaminase 5-Fluorocytosine Purinenucleoside 6-Methylpurine deoxyriboside; phosphorylase FludarabineUracil phosphoribosyl 5-Fluorocytosine; 5-Fluorouracil transferaseThymidylate kinase Azidothymidine

Desirably, the PCPV of the invention carries in its genome a suicidegene encoding a polypeptide having at least cytosine deaminase (CDase)activity. In the prokaryotes and lower eukaryotes (it is not present inmammals), CDase is involved in the pyrimidine metabolic pathway by whichexogenous cytosine is transformed into uracil by means of a hydrolyticdeamination. CDase also deaminates an analogue of cytosine, i.e.5-fluorocytosine (5-FC), thereby forming 5-fluorouracil (5-FU), acompound which is cytotoxic by itself but even more when it is convertedinto 5-fluoro-UMP (5-FUMP) by the action of uracil phosphoribosyltransferase (UPRTase).

CDase and UPRTase encoding nucleic acid can be obtained from anyprokaryotes and lower eukaryotes. Gene sequences and encoded enzymeshave been published and are available in specialized data banks such asSWISSPROT EMBL, Genbank, Medline and the like). Saccharomyces cerevisiaeCDase (FCY1 gene) and/or UPRTase (FUR1 gene) are preferred in thecontext of this invention (Kern et al., 1990, Gene, 88: 149-57).Functional variants of these genes may also be used. Such variantspreferably retain a degree of identity of at least 75%, with the aminoacid sequence of the native counterpart. A N-terminally truncatedUPRTase functional analogue is particularly useful in the context of theinvention due to its ability to exhibit a higher UPRTase activity thanthat of the native enzyme (e.g. as described in EP998568 with a deletionof the 35 first residues up to the second Met residue of the nativeprotein). Also preferred is a polypeptide having both CDase and UPRTaseactivities engineered by fusion of two enzymatic activities (see e.g.FCY1::FUR1 and FCY1::FUR1[Delta] 105 (FCU1) and FCU1-8 polypeptidesdescribed in WO96/16183, EP998568 and WO2005/07857). Of particularinterest is the FCU1 suicide gene (or FCY1::FUR1[Delta] 105 fusion)encoding a polypeptide comprising the amino acid sequence represented inthe sequence identifier SEQ ID NO: 1 of WO2009/065546).

Armament Gene Products

Other foreign nucleic acids may be used in the context of the inventionto arm the PCPV virus with the aim of potentiating anti-tumor efficacy.In one embodiment, the armed gene product is selected from the groupconsisting of nucleoside pool modulators (e.g., cytidine deaminase andnotably yeast cytidine deaminase (CDD1) or human cytidine deaminase(hCD); see EP16306831.5); polypeptides acting on metabolic and immunepathways (e.g., adenosine deaminase and notably the human adenosinedeaminase huADA1 or huADA2; see EP17306012.0); polypeptides acting onthe apoptotic pathway; endonucleases (like restriction enzymes,CRISPR/Cas9), and target-specific RNAs (e.g., miRNA, shRNA, siRNA).

Detectable Gene Products

Typically, such a polypeptide is detectable by spectroscopic,photochemical, biochemical, immunochemical, chemical, or other physicalmeans and thus may permit to identify the recombinant PCPV within a hostcell or subject. Non-limiting examples of suitable detectable geneproducts includes mCherry, Emerald, firefly luciferase and greenfluorescent proteins (GFP and enhanced version thereof e-GFP) detectableby fluorescent means as well as beta-galactosidase detectable bycolorimetric means.

The present invention also encompasses PCPV expressing two or morepolypeptides of interest as described herein, e.g. at least twoantigenic polypeptides (e.g. HPV-16 E6 and E7 polypeptides), at leastone antigen and one cytokine (e.g. MUC1 antigen and IL-2), at least twoantigens and one cytokine (e.g. HPV E6 and E7 antigens and IL-2), etc.

In the context of the present invention, the encoded polypeptide ofinterest for use herein may incorporate structural features which arebeneficial to its expression by the PCPV of the invention and itstherapeutic effect in the subject; such as ones permitting to facilitatecloning in the PCPV genome (modification of potential cleavage sites),to reinforce antigenic nature (modification of glycosylation sites)and/or to improve MHC class I and/or MHC class II presentation (e.g.membrane anchorage). Membrane anchorage can be achieved by incorporatingin the polypeptide of interest a membrane-anchoring sequence and asecretory sequence (i.e. a signal peptide) if the native polypeptidelacks it. Briefly, signal peptides usually comprise 15 to 35 essentiallyhydrophobic amino acids which are then removed by a specific ER(endoplasmic reticulum)-located endopeptidase to give the maturepolypeptide. Trans-membrane peptides are also highly hydrophobic innature and serve to anchor the polypeptides within cell membrane. Thechoice of the trans-membrane and/or signal peptides which can be used inthe context of the present invention is vast. They may be obtained fromcellular or viral polypeptides such as those of immunoglobulins, tissueplasminogen activator, insulin, rabies glycoprotein, the HIV virusenvelope glycoprotein or the measles virus F protein or may besynthetic. Preferably, the secretory sequence is inserted at theN-terminus of the polypeptide downstream of the codon for initiation oftranslation and the membrane-anchoring sequence at the C-terminus,preferably immediately upstream of the stop codon.

Generation and Expression of the Foreign Nucleic Acid(s)

The foreign nucleic acid(s) to be expressed by the PCPV of the presentinvention may be easily generated by a number of ways known to thoseskilled in the art (e.g. cloning, PCR amplification, DNA shuffling). Forexample, such a foreign nucleic acid can be isolated from any availablesource (e.g. biologic materials described in the art, cDNA and genomiclibraries, or any prior art vector known to include it) using sequencedata available to the skilled person (e.g. publications, patentapplications, Genbank, etc.) and then suitably inserted in the PCPVgenome. Alternatively, they can also be generated by chemical synthesisin automatized process (e.g. assembled from overlapping syntheticoligonucleotides or synthetic gene). Preferably, such a foreign nucleicacid is obtained from cDNA and does not comprise intronic sequences.Modification(s) can be generated by a number of ways known to thoseskilled in the art, such as chemical synthesis, site-directedmutagenesis, PCR mutagenesis, etc.

In the context of the present invention, the foreign nucleic acid can beoptimized for providing high level expression in a particular host cellor subject. It has been indeed observed that, the codon usage patternsof organisms are highly non-random and the use of codons may be markedlydifferent between different hosts. As the foreign nucleic acid might befrom prokaryote (e.g. bacterial or viral antigen) or lower eukaryoteorigin (e.g. suicide gene product), it may have an inappropriate codonusage pattern for efficient expression in higher eukaryotic cells (e.g.human). Typically, codon optimization is performed by replacing one ormore “native” codon corresponding to a codon infrequently used by one ormore codon encoding the same amino acid which is more frequently used inthe subject to treat. It is not necessary to replace all native codonscorresponding to infrequently used codons since increased expression canbe achieved even with partial replacement.

Further to optimization of the codon usage, expression can also beimproved through additional modifications of the foreign nucleic acid.For example, the nucleic acid sequence can be modified so as to preventclustering of rare, non-optimal codons being present in concentratedareas and/or to suppress or modify “negative” sequence elements whichare expected to negatively influence expression levels. Such negativesequence elements include without limitation the regions having veryhigh (>80%) or very low (<30%) GC content; AT-rich or GC-rich sequencestretches; unstable direct or inverted repeat sequences; R A secondarystructures; and/or internal cryptic regulatory elements such as internalTATA-boxes, chi-sites, ribosome entry sites, and/or splicingdonor/acceptor sites.

Moreover, when homologous foreign nucleic acids need to be expressed bythe PCPV of the invention, at least one of the homologous sequences (atleast portion thereof) can be degenerated over the full-length nucleicacid or portion(s) thereof so as to reduce sequence identity. It isindeed advisable to degenerate the portions of sequences that show ahigh degree (e.g. at least 70%) of sequence identity so as to avoidhomologous recombination problems during production process and theskilled person is capable of identifying such portions by sequencealignment. Examples of proper sequence degeneration applied to HPVantigens obtained from various serotypes (e.g. HPV-16 and HPV-18 E6and/or E7 antigens) can be found in WO2008/092854.

In one embodiment, the foreign nucleic acid(s) to be inserted in andexpressed by the PCPV of the invention is/are operably linked tosuitable regulatory elements for expression in a desired host cell orsubject.

As used herein, the term “regulatory elements” or “regulatory sequence”refers to any element that allows, contributes or modulates theexpression of the nucleic acid(s) in a given host cell or subject,including replication, duplication, transcription, splicing,translation, stability and/or transport of the nucleic acid(s) or itsderivative (i.e. m RNA). As used herein, “operably linked” means thatthe elements being linked are arranged so that they function in concertfor their intended purposes. For example, a promoter is operably linkedto a nucleic acid if the promoter effects transcription from thetranscription initiation to the terminator of said nucleic acid at leastin a permissive host cell.

It will be appreciated by those skilled in the art that the choice ofthe regulatory sequences can depend on factors such as the nucleicacid(s) itself, the level of expression desired, etc. The promoter is ofspecial importance. In the context of the invention, it can beconstitutive directing expression of the foreign nucleic acid in manytypes of cells or specific to certain types of cells or tissues orregulated according to the phase of a viral cycle (e.g. late,intermediate or early). One may also use promoters that are repressedduring the production step in response to specific events or exogenousfactors (e.g. specific components, temperature, etc), in order tooptimize production of the recombinant PCPV and circumvent potentialtoxicity of the expressed polypeptide(s).

Poxvirus promoters are particularly adapted for expression inrecombinant PCPV. In one embodiment, the foreign nucleic acid insertedin the PCPV genome is placed under the control of a poxvirus promoter,preferably, a vaccinia virus promoter and more preferably one selectedfrom the group consisting of the 7.5K, H5R, 11K7.5 (Erbs et al., 2008,Cancer Gene Ther. 15(1): 18-28), SE, TK, pB2R, p28, p11 and K1Lpromoter, synthetic promoters such as those described in Chakrabarti etal. (1997, Biotechniques 23: 1094-7; Hammond et al, 1997, J. VirolMethods 66: 135-8; and Kumar and Boyle, 1990, Virology 179: 151-8) andearly/late chimeric promoters.

Those skilled in the art will appreciate that the regulatory elementscontrolling the expression of the at least one foreign nucleic acid mayfurther comprise additional elements for proper initiation, regulationand/or termination of transcription (e.g. polyA transcriptiontermination sequences), mRNA transport (e.g. nuclear localizationsequences), translation (e.g. an initiator Met, tripartite leadersequences, etc.), processing (e.g. signal peptides, transmembraneanchorage sequences, etc) and purification steps (e.g. a tag).

Insertion within the PCPV Genome and Generation of the Recombinant PCPV

Insertion of the at least one foreign nucleic acid(s) (equipped withappropriate regulatory elements) in the PCPV genome is made byconventional means, either using appropriate restriction enzymes or,preferably by homologous recombination.

In another aspect, the present invention provides a method forgenerating the recombinant PCPV of the invention by homologousrecombination between a transfer plasmid comprising the foreign nucleicacid (with its regulatory elements) flanked in 5′ and 3′ with PCPVsequences respectively present upstream and downstream the insertionsite and a PCPV genome. In one embodiment, said method comprise a stepof generating said transfer plasmid (e.g. by conventional molecularbiology methods) and a step of introducing said transfer plasmid into asuitable host cell, notably together with a PCPV virus comprising theflanking sequence present in the transfer plasmid (e.g. a wild-type PCPVvirus). Preferably, the transfer plasmid is introduced into the hostcell by transfection and the PCPV virus by infection.

The size of each flanking PCPV sequence may vary. It is usually at least100 bp and at most 1500 bp, with a preference for approximately 150 to500 bp on each side of the foreign nucleic acid, advantageously from 180to 450 bp, preferably from 200 to 400 bp and more preferably from 250 to350 bp with a specific preference for approximately 300 bp on each sideof the foreign nucleic acid to be inserted.

Various sites of insertion may be considered in the PCPV genomeincluding, without any limitation, in a viral gene, with a preferencefor a non-essential viral gene, in an intergenic region, in a portion ofthe PCPV genome which does not encode gene products or in duplicatedlocus (genes or locus which occur in 2 or more copies in the nativevirus genome). Upon insertion of the foreign nucleic acid(s) into thePCPV genome according to the method of the invention, the viral locus atthe insertion site may be deleted at least partially. In one embodiment,this deletion or partial deletion may result in suppressed expression ofthe viral gene product encoded by the deleted PCPV sequence resulting ina defective PCPV virus for said virus function.

Examples of insertion sites are given in U.S. Pat. No. 6,365,393. In apreferred embodiment, the foreign nucleic acid(s) is/are inserted in anon-essential and duplicated locus of the PCPV genome with a preferencefor the VEGF locus (Rziha et al., 2000, J. Biotechnol. 83(1-2):137-145). It has to be noted that VEGF gene exists in 2 copies in thePCPV genome and the present invention contemplates either insertion inboth VEGF locus or in only one (leaving a copy intact for providingviral function). Preferably, the foreign nucleic acid is inserted inboth VEGF locus resulting in two copies of the foreign nucleic acidinserted in the PCPV genome.

In certain embodiments, the method of the present invention further usesa selection and/or a detectable gene to facilitate identification of therecombinant PCPV. In preferred embodiments, the transfer plasmid furthercomprises a selection marker with a specific preference for the GPT gene(encoding a guanine phosphoribosyl transferase) permitting growth in aselective medium (e.g. in the presence of mycophenolic acid, xanthineand hypoxanthine) or a detectable gene encoding a detectable geneproduct such as GFP, e-GFP or mCherry. In one embodiment, the transferplasmid is introduced into the host cell in the presence of anendonuclease capable of providing a double-stranded break in saidselection or detectable gene. Said endonuclease may be in the form of aprotein or expressed by an expression vector. Representative embodimentsare illustrated in the Example section.

Homologous recombination permitting to generate the recombinant PCPV ispreferably carried out in appropriate host cells (e.g. Bovine Turbinatecells).

Particularly preferred embodiments are methods for the generation of:

-   -   a PCPV virus comprising inserted within one or both VEGF locus a        nucleic acid encoding the human MUC-1 antigen (e.g. as described        in WO92/07000 and U.S. Pat. No. 5,861,381) preferably under the        transcriptional control of the early/late vaccinia pH5R        promoter; or    -   a PCPV virus comprising inserted within one or both VEGF locus a        nucleic acid encoding a membrane anchored HPV-16 non-oncogenic        E7 antigen (as described in WO99/03885) preferably under the        transcriptional control of the early/late vaccinia p7.5        promoter.

Production of PCPV Virus

In another aspect, the present invention relates to a method foramplifying the PCPV of the invention. Preferably said method comprisesthe steps of a) preparing a producer cell line, b) transfecting orinfecting the prepared producer cell line, c) culturing the transfectedor infected producer cell line under suitable conditions so as to allowthe production of the virus (e.g. infectious viral particles), d)recovering the produced virus from the culture of said producer cellline and optionally e) purifying said recovered virus.

In one embodiment, the producer cell is a mammalian (e.g. human ornon-human) cell and preferably HeLa cells (e.g. ATCC-CRM-CCL-2™ orATCC-CCL-2.2™).

Producer cells are preferably cultured in an appropriate medium whichcan, if needed, be supplemented with serum and/or suitable growthfactor(s) or not (e.g. a chemically defined medium preferably free fromanimal- or human-derived products). An appropriate medium may be easilyselected by those skilled in the art depending on the producer cells.Such media are commercially available. Producer cells are preferablycultivated at a temperature comprised between +30° C. and +38° C. (morepreferably at approximately 37° C.) for between 1 and 8 days beforeinfection. If needed, several passages of 1 to 8 days may be made inorder to increase the total number of cells.

In step b), producer cells are infected by the PCPV virus describedherein under appropriate conditions using an appropriate multiplicity ofinfection (MOI) to permit productive infection of producer cells. Forillustrative purposes, an appropriate MOI ranges from 10⁻³ to 50, with aspecific preference for a MOI comprises from 0.01 to 5. Infection stepis carried out in a medium which may be the same as or different fromthe medium used for the culture of producer cells.

In step c), infected producer cells are then cultured under appropriateconditions well known to those skilled in the art until progeny virus(e.g. infectious virus particles) is produced. Culture of infectedproducer cells is also preferably performed in a medium which may be thesame as or different from the medium/media used for culture of producercells and for infection step, at a temperature between +32° C. and +37°C., for 1 to 5 days.

In step d), the virus produced in step c) is collected from the culturesupernatant and/or the producer cells. Recovery from producer cells mayrequire a step allowing the disruption of the producer cell membrane toallow the liberation of the virus. The disruption of the producer cellmembrane can be induced by various techniques well known to thoseskilled in the art, including but not limited to: freeze/thaw, hypotoniclysis, sonication, microfluidization, high shear (also called highspeed) homogenization or high-pressure homogenization.

Viral vectors may then be further purified, using purification stepswell known in the art. Various purification steps can be envisaged,including clarification, enzymatic treatment (e.g. endonuclease,protease, etc), chromatographic and filtration steps. Appropriatemethods are described in the art (e.g. in WO2007/147528).

Pharmaceutical Composition

In an aspect is provided a composition comprising a therapeuticallyeffective amount of the PCPV described herein and a pharmaceuticallyacceptable vehicle. In embodiments, the PCPV is recombinant andcomprises inserted in its genome one or more foreign nucleic acid (withsuitable regulatory elements as described before), notably one or moreforeign nucleic acid encoding an antigenic polypeptide such as a cancerantigen, an antigen originating from a pathogenic organism, and/or animmunostimulatory polypeptide, etc). In specific embodiment, thecomposition can comprise exosomes generated after PCPV infection.

A “therapeutically effective amount” corresponds to the amount of PCPVthat is sufficient for producing beneficial results. Such atherapeutically effective amount may vary as a function of variousparameters such as the mode of administration; the age and weight of thesubject; the nature and extent of symptoms; the ability of the subjectto respond to the treatment, kind of concurrent treatment; the frequencyof treatment and/or the need for prevention or therapy, etc.

When “prophylactic” use is concerned, the composition is administered ata dose sufficient to prevent or to delay the onset and/or establishmentand/or relapse of the pathological condition, especially in a subject atrisk. For “therapeutic” use, the composition is administered to asubject diagnosed as having a disease or pathological condition with thegoal of treating it, optionally in association with one or moreconventional therapeutic modalities.

The term “pharmaceutically acceptable vehicle” is intended to includeany and all carriers, solvents, diluents, excipients, adjuvants,dispersion media, coatings, antibacterial and antifungal agents,absorption agents and the like compatible with administration in mammalsand in particular human subjects. Non-limiting examples ofpharmaceutically acceptable vehicles include water, NaCl, normal salinesolutions, lactated Ringers, saccharide solution (e.g. glucose,trehalose, saccharose, dextrose, etc) alcohols, oils, gelatins,carbohydrates such as lactose, amylose or starch, fatty acid esters,hydroxymethycellulose, and the like as well as other aqueousphysiologically balanced salt solutions may be used (see for example themost current edition of Remington: The Science and Practice of Pharmacy,A. Gennaro, Lippincott, Williams&Wilkins).

In one embodiment, the PCPV composition of the invention is formulatedappropriately to ensure its stability under the conditions ofmanufacture and long-term storage (i.e. for at least 6 months, with apreference for at least two years) at freezing (e.g. −70° C., −20° C.),refrigerated (e.g. 4° C.) or ambient (e.g. 20-25° C.) temperature. Suchformulations generally include a liquid carrier such as aqueoussolutions.

Advantageously, the formulation for use herein is suitably buffered forhuman use, preferably at physiological or slightly basic pH (e.g. fromapproximately pH 7 to approximately pH 9 with a specific preference fora pH comprised between 7 and 8 and more particularly close to 7.5).Suitable buffers include without limitation TRIS(tris(hydroxymethyl)methylamine), TRIS-HCl(tris(hydroxymethyl)methylamine-HCl), HEPES(4-2-hydroxyethyl-1-piperazineethanesulfonic acid), phosphate buffer(e.g. PBS), ACES (N-(2-Acetamido)-aminoethanesulfonic acid), PIPES(Piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPSO(3-(N-Morpholino)-2-hydroxypropanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), TES(2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), DIPSO(3-[bis(2-hydroxyethyl)amino]-2-hydroxypropane-1-sulfonic acid), MOBS(4-(N-morpholino)butanesulfonic acid), TAPSO(3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid),HEPPSO (4-(2-Hydroxyethyl)-piperazine-1-(2-hydroxy)-propanesulfonicacid), POPSO(2-hydroxy-3-[4-(2-hydroxy-3-sulfopropyl)piperazin-1-yl]propane-1-sulfonicacid), TEA (triethanolamine), EPPS(N-(2-Hydroxyethyl)-piperazine-N′-3-propanesulfonic acid), and TRICINE(N-[Tris(hydroxymethyl)-methyl]-glycine). Preferably, said buffer isselected from TRIS-HCl, TRIS, Tricine, HEPES and phosphate buffercomprising a mixture of Na₂HPO₄ and KH₂PO₄ or a mixture of Na₂HPO₄ andNaH₂PO₄. Said buffer (in particular those mentioned above and notablyTRIS-HCl) is preferably present in a concentration of 10 to 50 mM. Itmight be beneficial to also include in such formulations a monovalentsalt so as to ensure an appropriate osmotic pressure. Said monovalentsalt may notably be selected from NaCl and KCl, preferably saidmonovalent salt is NaCl, preferably in a concentration of 10 to 500 mM.

The formulation may also include a cryoprotectant so as to protect thevirus-based composition at low storage temperature. Suitablecryoprotectants include without limitation sucrose (or saccharose),trehalose, maltose, lactose, mannitol, sorbitol and glycerol, preferablyin a concentration of 0.5 to 20% (weight in g/volume in L, referred toas w/v). For example, sucrose is preferably present in a concentrationof 5 to 15% (w/v).

The PCPV composition, and especially a liquid composition thereof, mayfurther comprise a pharmaceutically acceptable chelating agent, and inparticular an agent chelating dications for improving stability. Thepharmaceutically acceptable chelating agent may notably be selected fromethylenediaminetetraacetic acid (EDTA),1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA),ethylene glycol tetraacetic acid (EGTA), dimercaptosuccinic acid (DMSA),diethylene triamine pentaacetic acid (DTPA), and2,3-Dimercapto-1-propanesulfonic acid (DMPS). The pharmaceuticallyacceptable chelating agent is preferably present in a concentration ofat least 50 μM with a specific preference for a concentration of 50 to1000 μM. Preferably, said pharmaceutically acceptable chelating agent isEDTA present in a concentration close to 150 μM.

Additional compounds may further be present to increase stability of thePCPV composition. Such additional compounds include, without limitation,C₂-C₃ alcohol (desirably in a concentration of 0.05 to 5% (volume/volumeor v/v)), sodium glutamate (desirably in a concentration lower than 10mM), non-ionic surfactant (U.S. Pat. No. 7,456,009, US2007-0161085) suchas Tween 80 (also known as polysorbate 80) at low concentration below0.1%. Divalent salts such as MgCl₂ or CaCl₂ have been found to inducestabilization of various biological products in the liquid state (seeEvans et al. 2004, J Pharm Sci. 93:2458-75 and U.S. Pat. No. 7,456,009).Amino acids, and in particular histidine, arginine or methionine, havebeen found to induce stabilization of various viruses in the liquidstate (see WO2016/087457).

The presence of high molecular weight polymers such as dextran orpolyvinylpyrrolidone (PVP) is particularly suited for freeze-driedcompositions, usually obtained by a process involving vacuum drying andfreeze-drying and the presence of these polymers assists in theformation of the cake during freeze-drying (see e.g. WO03/053463;WO2006/085082; WO2007/056847; WO2008/114021 and WO2014/053571).

In accordance with the present invention, formulation of the PCPVcomposition can also be adapted to the mode of administration to ensureproper distribution or delayed release in vivo. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylacticacid and polyethylene glycol. (see e.g. J. R. Robinson in “Sustained andControlled Release Drug Delivery Systems”, ed., Marcel Dekker, Inc., NewYork, 1978; WO01/23001; WO2006/93924; WO2009/53937).

For illustrative purposes, Tris-buffered formulations (Tris-HCl pH8)comprising saccharose 5% (w/v), sodium glutamate 10 mM, and NaCl 50 mMare adapted to the preservation of the composition of the invention from−20° C. to 5° C.

Dosage

The composition of the present invention can be formulated in individualdoses, each dose containing from about 10³ to 10¹² vp (viral particles),iu (infectious unit) or pfu (plaque-forming units) of a defined PCPVdepending on the quantitative technique used. The quantity of a definedPCPV present in a sample can be determined by routine titrationtechniques, e.g. by counting the number of plaques following infectionof permissive cells (e.g. HeLa cells) to obtain a plaque forming units(pfu) titer, by measuring the A260 absorbance (vp titers), or still byquantitative immunofluorescence, e.g. using anti-virus antibodies (iutiters). Further refinement of the calculations necessary to adapt theappropriate dosage for a subject or a group of subjects may be routinelymade by a practitioner, in the light of the relevant circumstances. As ageneral guidance, individual doses which are suitable for PCPVcomposition comprise from approximately 10³ to approximately 10¹² pfu,advantageously from approximately 10⁴ pfu to approximately 10¹¹ pfu,preferably from approximately 10⁵ pfu to approximately 10¹⁰ pfu; andmore preferably from approximately 10⁶ pfu to approximately 10⁹ pfu andnotably individual doses of approximately 10⁷, 5×10⁷, 10⁸ or 5×10⁸ pfuare particularly preferred.

Administration

Any of the conventional administration routes is applicable in thecontext of the invention including parenteral, topical or mucosalroutes. Parenteral routes are intended for administration as aninjection or infusion and encompass systemic as well as local routes.Parenteral injection types that may be used to administer the PCPVcomposition include intravenous (into a vein, such as the portal veinfeeding liver), intravascular (into a blood vessel), intra-arterial(into an artery such as hepatic artery), intradermal (into the dermis),subcutaneous (under the skin), intramuscular (into muscle),intraperitoneal (into the peritoneum) and intratumoral (into a tumor orits close vicinity) and also scarification. Administration can be in theform of a single bolus dose, or may be, for example, by a continuousperfusion pump. Mucosal administrations include without limitationoral/alimentary, intranasal, intratracheal, intrapulmonary, intravaginalor intra-rectal route. Topical administration can also be performedusing transdermal means (e.g. patch and the like). Preferably, the PCPVcomposition is formulated for intravenous, intramuscular, subcutaneousor intratumoral administration.

Administrations may use conventional syringes and needles (e.g.Quadrafuse injection needles) or any compound or device available in theart capable of facilitating or improving delivery of a virus in thesubject (e.g. electroporation for facilitating intramuscularadministration). An alternative is the use of a needleless injectiondevice (e.g. Biojector™ device). Transdermal patches may also beenvisaged.

The composition of the invention is suitable for a single administrationor a series of administrations. It is also possible to proceed viasequential cycles of administrations that are repeated after a restperiod. Intervals between each administration can be from three days tosix months (e.g. 24 h, 48 h, 72 h, weekly, every two weeks, monthly orquarterly, etc). Intervals can also be irregular. The doses can vary foreach administration within the range described above.

Therapeutic Use of the PCPV Composition and Method of Treatment

In another aspect, the invention relates to a recombinantpseudocowpoxvirus (PCPV) or a composition of the invention (inparticular a pharmaceutical composition), for use as a medicament, inparticular for treating or preventing diseases or pathological conditioncaused by a pathogenic organism or an unwanted cell division accordingto the modalities described herein, as well as to a method of treatmentcomprising administering the recombinant pseudocowpoxvirus (PCPV) or thecomposition of the invention to a subject in need thereof in an amountsufficient to treat or prevent such a disease or pathological condition.The invention also relates to the use of the recombinantpseudocowpoxvirus (PCPV) or the composition of the invention for themanufacture of a drug for treating or preventing diseases orpathological condition caused by a pathogenic organism or an unwantedcell division according to the modalities described herein. Theinvention also relates to the use of the recombinant pseudocowpoxvirus(PCPV) or the composition of the invention for treating or preventingdiseases or pathological condition caused by a pathogenic organism or anunwanted cell division according to the modalities described herein. Apreferred therapeutic scheme or treatment method involves 2 to 6 weeklyadministrations possibly followed by 2 to administrations at 3 weeksinterval of the PCPV composition comprising 10⁶ to 10⁹ pfu. In apreferred embodiment, the disease or pathological condition to betreated is a proliferative disease. Accordingly, the present inventionalso relates to a method for inhibiting tumor cell growth comprisingadministering the composition of the present invention to a subject inneed thereof. In the context of the invention, the methods and useaccording to the invention aim at slowing down, curing, ameliorating orcontrolling the occurrence or the progression of the targeted disease.

A “disease” (and any form of disease such as “disorder” or “pathologicalcondition” and the like) is typically characterized by identifiablesymptoms. Exemplary diseases include, but are not limited to, infectiousdiseases that result from an infection with a pathogenic organism (e.g.bacteria, parasite, virus, fungus, etc) and proliferative diseasesinvolving abnormal proliferation of cells. As used herein, the term“proliferative disease” encompasses any disease or pathologicalcondition resulting from uncontrolled cell growth and spread includingcancers and some cardiovascular diseases (e.g. restenosis that resultsfrom the proliferation of the smooth muscle cells of the blood vesselwall, etc.). The term “cancer” may be used interchangeably with any ofthe terms “tumor”, “malignancy”, “neoplasm”, etc. These terms are meantto include any type of tissue, organ or cell, any stage of malignancy(e.g. from a pre-lesion to stage IV) and encompass solid tumors andblood borne tumors as well as primary and metastatic cancers.

Representative examples of cancers that may be treated using thecomposition and methods of the invention include, without limitation,carcinoma, lymphoma, blastoma, sarcoma, and leukemia and moreparticularly bone cancer, gastrointestinal cancer, liver cancer,pancreatic cancer, gastric cancer, colorectal cancer, esophageal cancer,oro-pharyngeal cancer, laryngeal cancer, salivary gland carcinoma,thyroid cancer, lung cancer, cancer of the head or neck, skin cancer,squamous cell cancer, melanoma, uterine cancer, cervical cancer,endometrial carcinoma, vulvar cancer, ovarian cancer, breast cancer,prostate cancer, cancer of the endocrine system, sarcoma of soft tissue,bladder cancer, kidney cancer, glioblastoma and various types of thecentral nervous system (CNS), etc. In one embodiment the methods and useaccording to the present invention is for treating a cancer selectedfrom the group consisting of renal cancer (e.g. clear cell carcinoma),prostate cancer (e.g. hormone refractory prostate adenocarcinoma),breast cancer (e.g. metastatic breast cancer), colorectal cancer, lungcancer (e.g. non-small cell lung cancer) liver cancer (e.g.hepatocarcinoma), gastric cancer, bile duct carcinoma, endometrialcancer, pancreatic cancer and ovarian cancer cancers and a cancer thatoverexpresses MUC1. Preferred embodiments are directed to a PVPCencoding the MUC1 antigen as described herein for use for treating asubject having a non-small cell lung cancer (NSCL) and a PVPC encodingthe HPV16 E7 antigen as described herein for use for treating a subjecthaving a HPV-positive cancer such as a cervix cancer or a head and neckcancer.

Representative examples of infectious diseases that may be treated usingthe composition and methods of the invention include, withoutlimitation, a) viral diseases such as those resulting from infection byan herpes virus (HSV1, HSV2, or VZV), a papillomavirus (HPV), a poxviruscausing variola or chicken pox, an enterovirus, a retrovirus such as HIVcausing AIDS, a cytomegalovirus, a flavivirus (e.g. causing Japaneseencephalitis, hepatitis C, dengue and yellow fever), an Hepadnavirus(e.g. HBV), an orthomyxovirus (e.g. influenza virus), a paramyxovirus(e.g. parainfluenzavirus, mumps virus, measles virus and respiratorysyncytial virus (RSV)), a coronavirus (e.g. SARS), rhabdovirus androtavirus; b) diseases resulting from infection by bacteria, forexample, Escherichia, Enterobacter, Salmonella, Staphylococcus,Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus,Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus,Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium,Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella,Yersinia, Haemophilus, or Bordetella; and (c) fungal diseases includingbut not limited to candidiasis, aspergillosis, histoplasmosis,cryptococcal meningitis; and d) parasitic diseases including but notlimited to malaria, Pneumocystis carnii pneumonia, leishmaniasis,cryptosporidiosis, toxoplasmosis, and trypanosome infection.

Typically, upon administration according to the modalities describedherein, the composition of the invention provides a therapeutic benefitto the treated subject which can be evidenced by an observableimprovement of the clinical status over the baseline status or over theexpected status if not treated. An improvement of the clinical statuscan be easily assessed by any relevant clinical measurement typicallyused by physicians or other skilled healthcare staff. In the context ofthe invention, the therapeutic benefit can be transient (for one or acouple of months after cessation of administration) or sustained (forseveral months or years). As the natural course of clinical status whichmay vary considerably from a subject to another, it is not required thatthe therapeutic benefit be observed in each subject treated but in asignificant number of subjects (e.g. statistically significantdifferences between two groups can be determined by any statistical testknown in the art, such as a Tukey parametric test, the Kruskal-Wallistest the U test according to Mann and Whitney, the Student's t-test, theWilcoxon test, etc).

When the method or use of the invention is aimed at treating aproliferative disease, in particular cancer, a therapeutic benefit canbe evidenced at least temporarily by for instance a reduction in thetumor number; a reduction of the tumor size, a reduction in the numberor extent of metastases, an increase in the length of remission, astabilization (i.e. not worsening) of the state of disease, a delay orslowing disease progression or severity, a prolonged survival, a betterresponse to the standard treatment, an improvement of quality of life, areduced mortality, etc. “Treating” can also mean prolonging survival ofa subject beyond that expected in the absence of treatment.

When the method or use of the invention is aimed at treating aninfectious disease, a therapeutic benefit can be evidenced by forinstance, a decrease of the amount of the infecting pathogenic organismquantified in blood, plasma, or sera of a treated subject, and/or astabilized (not worsening) state of the infectious disease (e.g.stabilization of conditions typically associated with the infectiousdisease such as inflammatory status), and/or the reduction of the levelof specific seric markers (e.g. decrease of alanine aminotransferase(ALT) and/or aspartate aminotransferase (AST) associated with liver poorcondition usually observed in chronic hepatitis C), decrease in thelevel of any antigen associated with the occurrence of an infectiousdisease and/or the appearance or the modification of the level ofantibodies to the pathogenic organism and/or an improved response of thetreated subject to conventional therapies (e.g. antibiotics) and/or asurvival extension as compared to expected survival if not receiving thecomposition treatment.

The appropriate measurements such as blood tests, analysis of biologicalfluids and biopsies as well as medical imaging techniques can be used toassess a clinical benefit. They can be performed before theadministration (baseline) and at various time points during treatmentand after cessation of the treatment. For general guidance, suchmeasurements are evaluated routinely in medical laboratories andhospitals and a large number of kits are available commercially (e.g.immunoassays, quantitative PCR assays).

In another aspect, the composition of the invention is used oradministered for eliciting or stimulating and/or redirecting an immuneresponse in the treated subject. Accordingly, the present invention alsoencompasses a method for eliciting or stimulating and/or re-orienting animmune response (e.g. to tumor or infected cells) comprisingadministering the composition of the invention to a subject in needthereof, in an amount sufficient according to the modalities describedherein so as to activate the subject's immunity. The elicited,stimulated or redirected immune response can be specific (i.e. directedto epitopes/antigens) and/or non-specific (innate), humoral and/orcellular, notably a CD4+ or CD8+-mediated T cell response. The abilityof the composition described herein to elicit, stimulate or redirect animmune response can be evaluated either in vitro (e.g. using biologicalsamples collected from the subject) or in vivo using a variety of director indirect assays which are standard in the art (see for exampleColigan et al., 1992 and 1994, Current Protocols in Immunology; ed JWiley & Sons Inc, National Institute of Health or subsequent editions).Those cited above in connection with the antigenic nature of apolypeptide are also appropriate.

In preferred embodiments, the method according to the invention, resultsin at least one the following properties:

-   -   The secretion of high levels of IFN-alpha from PBMC, preferably        at levels at least 50-fold higher than the levels observed        following administration of a MVA under the same conditions or        at levels at least 10 fold higher than the levels observed        following administration of a ORFV under the same condition;    -   The activation of monocyte-derived dendritic cells (e.g. can be        reflected by the induction of the activation marker CD86,        preferably at levels at least twice the levels observed        following administration of a MVA under the same conditions);    -   The induction of T cell activation or proliferation (e.g. as        indirectly reflected by highly granzyme B+ T cells);    -   A better cytokine/chemokine profile in MDSC (e.g. can be        reflected by higher levels of at least one of IFN-alpha and        MIP1alpha secreted by MDSC following infection of PCPV than        after infection with MVA);    -   activation of APC (e.g. can be reflected by the upregulation of        CD86 in human macrophages following administration of PCPV);    -   a M2 to M1 conversion of human macrophages (e.g. can be        reflected by higher secretion of IL-18, IL-6 and IP-10 in human        macrophages after administration of PCPV than after infection        with MVA; and/or    -   The induction of immunity through a TLR9-mediated pathway or        other innate immunity-stimulating pathways such as cGAS-STING        and RIG-I-like receptors (RLRs) (e.g., Retinoic acid-inducible        gene I (RIG-I) and Melanoma differentiation-associated gene 5        (MDA5)).

Accordingly, the present invention also encompasses methods forincreasing secretion of IFN-alpha from an immune cell such as PBMC, forinducing the activation of monocyte-derived dendritic cells, forinducing activation and/or proliferation of T cell, for inducing M2 toM1 conversion of human macrophages, for reducing MDSC-associated immunesuppression and/or for reducing immune suppression, any of such methodscomprising administering the composition of the invention to a subjectin need thereof (in vivo method) or contacting a precursor cell with thecomposition of the invention (in vitro method) so as to achieve thedesired result(s).

In one embodiment, the composition or methods of the invention can beused or carried out in conjunction with one or more other therapeuticagents which are available for treating or preventing the disease orpathological condition to be treated or prevented. In one embodiment theother therapeutic agent is selected from the group consisting ofsurgery, radiotherapy, chemotherapy, cryotherapy, hormonal therapy,toxin therapy, immunotherapy, cytokine therapy, targeted cancer therapy,gene therapy, photodynamic therapy and transplantation, etc. A method oruse according to the invention may include a third or even furthertherapeutic agent.

Such additional anticancer therapy/ies is/are administered to thesubject in accordance with standard practice before, after, essentiallyconcurrently or in an interspersed manner with the PCPV composition ofthe present invention. Essentially concurrent administration of two ormore therapeutic agents does not require that the agents be administeredat the same time or by the same route, as long as there is an overlap inthe time period during which the agents are exerting their therapeuticeffect. Concurrent administration includes administering the compositionof the invention within the same day (e.g. 0.5, 1, 2, 4, 6, 8, 10, 12hours) as the other therapeutic agent. Although any order iscontemplated by the present invention, it is preferred that thecomposition of the invention be administered to the subject before theother therapeutic agent.

In specific embodiments, the method or use according to the inventionmay be carried out in conjunction with surgery. For example, thecomposition may be administered after partial or total surgicalresection of a tumor (e.g. by local application within the excised zone,for example).

In other embodiments, the method or use according to the invention canbe used in association with radiotherapy. Those skilled in the art canreadily formulate appropriate radiation therapy protocols and parameters(see for example Perez and Brady, 1992, Principles and Practice ofRadiation Oncology, 2nd Ed. JB Lippincott Co; using appropriateadaptations and modifications as will be readily apparent to thoseskilled in the field). The types of radiation that may be used notablyin cancer treatment are well known in the art and include electronbeams, high-energy photons from a linear accelerator or from radioactivesources such as cobalt or cesium, protons, and neutrons. Dosage rangesfor radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells. Regular X-rays doses for prolonged periods of time(3 to 6 weeks), or high single doses are contemplated by the presentinvention.

In certain embodiments of the invention, the composition of theinvention may be used in conjunction with chemotherapy. Representativeexamples of suitable chemotherapy agents currently available fortreating cancer include, without limitation, alkylating agents,topoisomerase I inhibitors, topoisomerase II inhibitors, platinumderivatives, inhibitors of tyrosine kinase receptors, cyclophosphamides,antimetabolites, DNA damaging agents and antimitotic agents.Representative examples of suitable chemotherapy agents currentlyavailable for treating infectious diseases include among otherantibiotics, antimetabolites, antimitotics and antiviral drugs (e.g.interferon alpha).

In further embodiments, the composition of the invention may be used inconjunction with immunotherapeutics such as anti-neoplastic antibodiesas well as siRNA and antisense polynucleotides. Representative examplesinclude among others monoclonal antibodies blocking specific immunecheckpoints such as anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-LAG3,anti-OX40 etc (e.g. Ipilimumab, tremelimumab pembrolizumab, nivolumab,pidilizumab, durvalumab. daclizumab, avelumab, atezolizumab, etc.,),monoclonal antibodies blocking Epidermal Growth Factor Receptor (inparticular cetuximab, panitumumab, zalutumumab, nimotuzumab, matuzumab,trastuzumab (Herceptin™), etc.,) and monoclonal antibodies blockingVascular Endothelial Growth Factor (in particular bevacizumab andranibizumab).

In still further embodiments, the composition of the present inventionmay be used in conjunction with adjuvant. Representative examples ofsuitable adjuvants include, without limitation, TLR3 ligands(Claudepierre et al., 2014, J. Virol. 88(10): 5242-55), TLR9 ligands(e.g. CpGs such as ODN1826 (Fend et al., 2014, Cancer Immunol. Res. 2,1163-74) and Litenimod (Li28) (Carpentier et al., 2003, Frontiers inBioscience 8, e115-127; Carpentier et al., 2006, Neuro-Oncology 8(1):60-6; EP 1 162 982; U.S. Pat. Nos. 7,700,569 and 7,108,844) and PDE5inhibitors such as sildenafil (U.S. Pat. Nos. 5,250,534, 6,469,012 andEP 463 756).

In additional embodiments, the composition or methods of the inventionmay be used according to a prime boost approach which comprisessequential administrations of a priming composition(s) and a boostingcomposition(s). Typically, the priming and the boosting compositions usedifferent vectors which comprise or encode at least an antigenic domainin common. Moreover, the priming and boosting compositions can beadministered at the same site or at alternative sites by the same routeor by different routes of administration. A preferred prime boostapproach in the context of the invention involves a PCPV composition asdescribed herein and a MVA composition encoding the same polypeptide ofinterest (e.g. the same antigen). In one embodiment, the MVA is used forpriming and the PCPV composition of the invention is used for boostingor vice versa (PCPV for priming and MVA for boosting). The presentinvention encompasses one or several administration(s) of the primingand/or the boosting composition(s) with a preference for subcutaneous,intratumoral and intravenous routes. A particularly preferred primeboost regimen comprises a PCPV priming composition administered byintratumoral route and a MVA boosting composition administered byintravenous route. In a preferred embodiment, the PCPV and MVAcomposition encodes the same tumor-associated antigen. In anotherembodiment, the period of time separating the administrations of thepriming and the boosting varies from one week to 6 months, with apreference for one week to one month and even more for a period of oneto two weeks. Individual doses of approximately 10⁶ pfu to approximately10⁹ pfu are particularly adapted for priming and boosting compositionsand notably individual doses of approximately 10⁶, 5×10⁶ 10⁷, 5×10⁷, 10⁸or 5×10⁸ pfu.

In another aspect, the present invention also provides a bovine papularstomatitis virus (BPSV), preferably a recombinant BPSV comprising atleast one foreign nucleic acid inserted in its genome. The term “bovinepapular stomatitis virus” or “BPSV” is used herein according to itsplain ordinary meaning within Virology and refers to a member of thePoxviridae family belonging to the Parapoxvirus genus.

The foreign nucleic acid(s) to be expressed by the recombinant BPSVis/are similar to those described in connection with PCPV as well as theregulatory elements required for expression in a desired host cell orsubject. Insertion site(s) of the foreign nucleic acid(s) within theBPSV genome and method of producing recombinant BPSV are also within thereach of the person skilled in the art. Method for amplifying BPSV isroutine based on the description given herein for the PCPV virus andgeneral knowledge, although producer cell lines, MOI, culture medium,etc can be adapted to the BPSV virus by the skilled artisan.

Composition comprising a therapeutically effective amount of the BPSVand a pharmaceutically acceptable vehicle are also part of thisinvention. Suitable dosages, administration routes and therapeutic usesare as described above for PCPV-comprising compositions.

All of the above cited disclosures of patents, publications and databaseentries are specifically incorporated herein by reference in theirentirety. Other features, objects, and advantages of the invention willbe apparent from the description and drawings and from the claims. Thefollowing examples are incorporated to demonstrate preferred embodimentsof the invention. However, in light of the present disclosure, thoseskilled in the art should appreciate that changes can be made in thespecific embodiments that are disclosed without departing from thespirit and scope of the invention.

EXAMPLES

Several viruses and strains have been developed to express tumorantigens and cytokines, and some of them are in advanced clinicaltrials. However, novel viral strains with improved immunogenicproperties are sought. In this perspective, we screened Parapoxvirusessuch as Pseudocowpox (PCPV) and Parapoxvirus Ovis (ORF) and comparedthem to a variety of other poxviruses. The comparison was conducted invitro with human primary immune cells, and in vivo with syngeneic mousetumor models.

Example 1: Profile of Secreted Cytokines/Chemokines in Infected HumanPeripheral Blood Mononuclear Cells (PBMCs)

Human peripheral blood mononuclear cells (PBMCs) were isolated fromwhole human blood of two healthy donors (EFS, Etablissement deStrasbourg; donor A and B). PBMCs were isolated by density gradientcentrifugation using Ficoll-Paque PLUS (GE HealthCare) and BloodSeparation Tubes (Greiner) and stored in liquid nitrogen.

Frozen aliquots of PBMCs were thawed in RPMI supplemented with 10% Fetalcalf serum (FSC) and dispatched in 24 well plates (5·10⁵ cells in 500μL/well). After 5 to 6 hours, cells were infected with the differentviruses (see below) at multiplicities of infection (MOI) between 10⁻³and 10. After overnight incubation (16 hours), cells were scraped, cellsand supernatant were transferred in Eppendorf tubes and centrifuged 10min at 300 g. Supernatant was isolated and frozen at −80° C. Cytokineand chemokine profiles were quantified by a Multiplex approach in thesupernatants using a Procartaplex 20plex inflammation panel(EPX200-12185-901). The analysis was carried out according to themanufacturer's recommendation using a MagPix device.

The different viruses tested are Parapoxviruses pseudocowpoxvirus (PCPVand Parapoxvirus ovis (ORF), as well as MVA, cowpox virus (CPX),Copenhagen vaccinia virus (Copwt), fowlpox (FPV), Myxomavirus (MYXV),Swine pox (SWPV), raccoonpoxvirus (RCN), Cotia virus (CTV) and Yaba-likedisease virus (YLDV). The viruses were obtained from the ATCCcollection, respectively ATCC-VR-644 (PCPV TJS), ATCC VR-1548 (ORFVNZ2), ATCC VR-302 (CPX Brighton), ATCC VR-115 (MYXV Lausanne), ATCCVR-363 (SWPX Kasza), ATCC VR-838 (RCN Herman), ATCC VR-464 (CTV SP AN32) and ATCC VR-937 (YLDV Davis); except MVA for which an empty MVAvector named MVAN33 was used (Genbank accession number EF675191), theCopenhagen vaccinia virus (see e.g. WO2009/065546) and FPV (see e.g.Laidlaw and Skinner, 2004; J. Gen. Virol., 85: 305-22).

As shown in FIG. 1, PCPV-infected PBMCs induced the secretion of veryhigh levels of IFN-alpha in a MOI-dependent way. Although the levelssecreted by the PCPV-infected PBMC vary between the two experiments,they are well above the moderate secretion levels of IFN-alpha observedwith SWPX and ORFV (another parapoxvirus). Compared with MVA, PCPVinduced a 1000-fold higher expression of IFN-alpha in human PBMCswhereas SWPX and ORF displayed a lower 10 to 100-fold induction.Copenhagen VV and other oncolytic vectors (e.g. RCN, RPV, CTV, CPX andMYX) did not raise the IFN alpha level.

Viability studies upon viral infection were carried out. Cells werestained with LiveDead “NearIR” and analyzed by flow cytometry(MacsQuant) to determine the proportion of dead and living cells. Theresults showed that ORFV and YLDV were particularly toxic: 90% of cellsinfected at the MOI of 5 died within 16 hours, while at least 50% ofliving cells were observed for all other viruses, including PCPV.

Example 2: Effect of PCPV on Human Monocyte-Derived Dendritic Cells, M2Macrophages and Myeloid-Derived Suppressor Cells (MDSCs)

Type I interferons like IFN-alpha are key players in immunotherapy ofcancer as well as altered immunosuppressive tumor environment andimproved adaptive anti-tumor responses against vaccine encoded and/ortumor-presented antigens as described e.g. by Parker et al. (2016, NatRev Cancer 16(3): 131-44) and Zitvogel et al. (2015, Nat Rev Immunol.15(7): 405-14) To address these aspects, we looked in vitro in humanprimary immune cells at the activation of human monocyte-deriveddendritic cells (moDC) and at the effect on immunosuppressive cellpopulations (re-programming of immunosuppressive cells like M2macrophages and MDSCs) upon PCPV treatment. Preclinical studies oninnate and adaptive immunity were also probed in murine syngeneic tumormodels.

Stimulation of moDCs.

To probe the activation of antigen-presenting cells (APCs), we workedwith monocyte-derived dendritic cells (moDCs). To obtains these cells,human monocytes were enriched by depletion from PBMCs (Miltenyi,Monocyte Isolation kit II). Monocytes were differentiated in dendriticcells (moDCs) by incubation in granulocyte-macrophage colony-stimulatingfactor (GM-CSF 20 ng/ml) and IL-4 (10 ng/ml) for 3 days. For stimulationassays, moDCs from three different donors (donor 12Jul16, donor 24Aug16and donor 31Aug16) were plated in 24-well plates and infected withMVAN33.1 or PCPV at the MOIs of 0.03, 0.3 and 1. The day after, thematuration marker CD86 was quantified by flow cytometry. Expressionlevels were measured by staining with anti-CD86-PE quantification of thecell-bound signal by flow cytometry

As illustrated in FIG. 2, PCPV-treated moDCs showed much higherexpression levels of the activation marker CD86 compared toMVAN33.1-treated cells. The beneficial effect on moDC stimulation wasseen at low MOI (0.03) and increased at MOI 0.3 and 3. As expected, nomoDC stimulation was detected upon mock treatment. Therefore, itappeared that PCPV infection increased CD86 expression on all donors atall MOI to higher levels than MVAN33. Copwt had no effect on moDC CD86expression (data not shown).

Effects on M2 Macrophages

Next, we addressed the question whether and to which extentimmunosuppressive cell types could be altered/re-educated by PCPV.

We generated in vitro derived CD163⁺CD206⁺ “M2-type” macrophagesaccording to a protocol adapted from Mia et al. (2014, Scand. J.Immunol. 79(5): 305-14). Briefly, after isolation from PBMCs obtainedfrom healthy donors either by positive CD14⁺ selection or by negativeselection using monocyte isolation kit II (Miltenyi, Biotec), 4×10⁵monocytes were cultured on 48 well plates in 500 μl Macrophage BaseMedium DXF (Promocell) supplemented with 50 ng/ml M-CSF (MiltenyiBiotec). Medium was changed at day 3 and day 7. At day 7,CD16^(hi)CD68⁺CD11b⁺ M0 macrophages were polarized towards a M2phenotype adding IL-4, IL-10 and TGF-beta (Miltenyi Biotec) to aconcentration of 20 ng/ml. Two days later, CD163⁺CD206⁺ M2 macrophageswere incubated with MVAN33.1 or PCPV at a MOI of 5, 1 and 0.3 and thesecretion of IL-18, IL-6, IP-10 and CD86 was assayed by flow cytometryor Luminex analysis. For this purpose, cells were incubated for 20 minwith human FcR Blocking Reagent (Miltenyi Biotec) and APC-labeledanti-CD86 (clone FM95, Miltenyi Biotec). Dead cells were labeled byautomatic propidium iodide staining before cell acquisitions recordedusing MACSQuant cytometer (Miltenyi). Data were analyzed using Kaluzasoftware (Beckman Coulter). Cytokines levels in the supernatant ofstimulated cells were quantified by Luminex analysis (ProcartaPlex,eBiosciences) on a MAGPIX device (Luminex XMAP Technologies).

As illustrated in FIG. 3, compared to MVA, PCPV augmented the secretionof IL-18 at MOI 5 and 1, the secretion of IL-6 and IP-10 at MOI 5. Thus,it appeared that PCPV changes the phenotype of M2 macrophages towards aM1-type phenotype. M2 macrophages are associated with a negative outcomein human NSCLC, while M1 macrophages represent a positive prognosticmarker (Yuan et al, 2015, Nature, Scientific Reports). These datasuggest a conversion to a less suppressive phenotype after PCPVtreatment compared to MVA treatment.

Effects on Myeloid-Derived Suppressor Cells MDSCs

Autologous CD14⁺ monocytes and CD8⁺ T cells were isolated from healthydonors by magnetic beads technology (Miltenyi). Two models wereinvestigated. MDSCs were generated from monocytes either by incubationwith 20 ng/ml GM-CSF, 10 ng/ml IL-4 and 1 μM prostaglandin E2, (FIG. 4)or in the presence of 10 ng/ml GM-CSF and 10 ng/ml IL-6 (FIG. 5) for 6to 7 days. The resulting MDSCs were cocultured with autologousCFSE-labeled CD8⁺ T cells in the presence of T cell activation beadscoated with anti-CD2, anti-CD3 and anti-CD28 or TransAct T CellReagent+IL-2 (Miltenyi Biotec). After 4-5 days, proliferation of T cellsand expression of Granzyme B in proliferating T cells were measured byflow cytometry. In addition, MDSCs alone were treated with PCPV, thenext day supernatant was taken to quantify analytes like cytokine andchemokines. As shown in FIG. 4, proliferation of activated T cells wasinhibited by co-cultured in vitro-derived MDSCs (see uninfected controlsin FIG. 4A; MDSC/T ratio of ½). However, the suppressive activityprovided by MDSCs on CTL proliferation can be inhibited after treatmentwith PCPV added to the co-culture as evidenced by the ability of PCPV torestore CTL proliferation at increasing MOIs (FIG. 4A) correlating withupregulation of Granzyme B production in co-cultures treated with PCPV(FIG. 4B). To be highlighted that the anti-suppressive effect in thepresence of PCPV was detected even at low MOI. These observationcorrelate with IFN alpha secretion measured in MDSCs cultures the dayafter treatment with PCPV (FIG. 4C).

As above, and as shown in FIG. 5 illustrating the GM-CSF, IL-6-derivedMDSC model, proliferation of activated T cells was strongly inhibited byco-cultured in vitro-derived MDSCs. Proliferation was partly restored,and Granzyme B expression was upregulated when co-cultures were treatedwith PCPV at increasing MOIs.

In summary, PCPV was shown to be superior than MVA and VV to trigger theexpression of CD86 in primary moDCs. Furthermore, PCPV treatmentincreased CD86 expression in human in vitro-derived CD163⁺CD206⁺“M2”-type macrophages, suggesting a shift to an antigen-presentingphenotype. In these cells, PCPV increased significantly the secretion ofIL-18, IL-6 and IP-10, signing a conversion towards a less suppressivemacrophage phenotype. The mode of action of PCPV on MDSCs could bematuration to a less suppressive phenotype or toxic effects of PCPV onMDSCs.

Example 3: Construction of Recombinant PCPV and Anti-Tumor Properties

The interest for PCPV as viral backbone was evaluated by generatingrecombinant PCPV encoding tumor antigens and testing them in tumorcontrol experiments.

Recombinant PCPV vectors were constructed from the wild-type strain TJS(ATTC, VR-634). PCPV, as all the Parapoxvirus, lack genes present orconserved in other poxviruses. These comprises homologues of mostpoxviral genes likely involved in nucleotide metabolism, includinghomologues of ribonucleotide reductase (RR), thymidine kinase (TK),guanylate kinase and thymidylate kinase. Therefore, the locus TKgenerally used for generation of recombinant vaccinia virus could not beused for introduction of recombinant gene in PCPV. However, it was shownthat recombinant ORF virus could be generated by insertion of thetransgene in the non-essential VEGF gene (Rziha et al., 2000, J.Biotechnol. 83(1-2): 137-145). This locus was therefore evaluated forthe generation of recombinant PCPV. It should be noticed that the VEGFgene is present in two copies in the PCPV genome on the left and rightgenome terminus.

A transfer plasmid was generated by cloning in pUC18 plasmid (availablee.g. in Thermo Fisher Scientific) two sequences of about 300 bp flankingthe PCPV VEGF gene. The sequences upstream the VEGF gene correspond tonucleotide position 6391 to 6089 or 138899 to 139201 of PCPV (GenbankNC_013804). The sequences downstream the VEGF gene correspond tonucleotide position 5545 to 5225 or 139745 to 140065 of PCPV. Thesesequences will allow homologous recombination between the transferplasmid and PCPV virus leading to recombinant virus with the foreignnucleic acid inserted in both VEGF locus.

Generation of PCP-GFP (PCPTG19106)

The selection cassette (eGFP/GPT), a fusion of the gene encoding theenhanced green fluorescent protein (eGFP) and the gene encoding guaninephosphoribosyl transferase (GPT) (see e.g. WO2009/065546) was positionedunder the control of p11K7.5 vaccinia promoter and inserted in thetransfer plasmid leading to pTG19106.

Generation of PCPTG19106 was performed by homologous recombination inBovine Turbinate cells (BT, ATCC CRL-1390) infected with PCPV andtransfected by nucleofection with pTG19106 (according to AmaxaNucleofector technology). Fluorescent and selective (GPT+) plaques wereselected. More specifically, GPT⁺ recombinant PCPV have a growthadvantage in a selective medium (mycophenolic acid, xanthine, andhypoxanthine), by maintaining the GPT selection marker during theisolation of recombinant PCPV. Recombinant virus was isolated fromGFP-fluorescent plaques and submitted to additional plaques purificationin BT cells. Virus structure and absence of parental PCPV was confirmedby multiple PCRs and DNA sequencing of the expression cassette. Theresulting virus PCPTG19106 was amplified in BT cells. Virus stocks weretitrated on BT cells by plaque assay.

Generation of PCP-m Cherry (PCPTG19153)

The sequence coding for the mCherry fluorescent protein was positionedunder the control of pH5R vaccinia promoter and inserted in the transferplasmid leading to pTG19153.

Generation of PCPTG19153 was performed by homologous recombination in BTcells infected with PCPTG19106 and transfected by nucleofection withpTG19153. Recombinant virus was isolated by selected mCherry-fluorescentplaques and submitted to additional plaques purification in BT cells.Virus structure and absence of parental PCPTG19106 was confirmed bymultiple PCRs and DNA sequencing of the expression cassette. Theresulting virus PCPTG19153 was amplified in BT cells.

Generation of PCP-HPV16E7 (PCPTG19178)

The sequence coding for SR-E7*™ (WO99/03885) was positioned under thecontrol of p7.5K promoter and inserted in the transfer plasmid leadingto pTG19178. Generation of PCPTG19178 was performed by homologousrecombination in BT cells infected with PCPTG19106 and transfected bynucleofection with pTG19178 in presence of an endonuclease generatingdouble-strand break in the GFP gene. Recombinant virus was isolated byselected GFP-fluorescent negative plaques and submitted to additionalplaques purification in BT cells. Virus structure and absence ofparental PCPTG19106 was confirmed by multiple PCRs and DNA sequencing ofthe expression cassette. The resulting virus PCPTG19178 was amplified inBT cells.

Production of pre-clinical batch of PCPTG19178 was performed by infectedHela cells (ATCC® CCL-2™) in several F500 flasks. Viral amplificationwas performed at 34 to 37° C., 5% CO2 for 72 h. Infected cells andmedium were then pelleted and frozen. The crude harvest was disruptedusing high shear homogenizer (SILVERSON L4R) and submitted to apurification process (e.g. as described in WO2007/147528). Briefly, thelysed viral preparation can be clarified by filtration, and purified bya tangential flow filtration (TFF) step. Purified virus was resuspendedin a suitable virus formulation buffer (e.g. 5% (w/v) Saccharose, 50 mMNaCl, 10 mM Tris/HCl, 10 mM Sodium Glutamate, pH8).

Generation of PCP-MUC1 (PCPTG19194)

The sequence coding for Muc1 (U.S. Pat. No. 5,861,381) was positionedunder the control of pH5R promoter and inserted in the transfer plasmidleading to pTG19194. Generation of PCPTG19194 was performed byhomologous recombination in BT cells infected with PCPTG19153 andtransfected by nucleofection with pTG19106 in presence of anendonuclease generating double-strand break in the m-Cherry gene.Recombinant virus was isolated by selected mCherry-fluorescent negativeplaques and submitted to additional plaques purification in BT cells.Virus structure and absence of parental PCPTG19194 was confirmed bymultiple PCRs and DNA sequencing of the expression cassette. Theresulting virus PCPTG19194 was amplified in BT cells. Pre-clinical batchwere produced in Hela cells as described above.

Generation of PCP-HBV (PCPTG19179)

The sequence coding for a fusion of HBV core Pol and env antigens (asdescribed in WO2013/007772) was positioned under the control of p7.5Kpromoter and inserted in the transfer plasmid leading to pTG19179.Generation of PCPTG19179 was performed by homologous recombination in BTcells infected with PCPTG19106 and transfected by nucleofection withpTG19179 in presence of an endonuclease generating double-strand breakin the GFP gene. Recombinant virus was isolated by selectedGFP-fluorescent negative plaques and submitted to additional plaquespurification in BT cells. Virus structure and absence of parental viruswas confirmed by multiple PCRs and DNA sequencing of the expressioncassette.

Tumor Control Experiments in Syngeneic MC38 Model:

The capacity of PCPV to control tumor growth and to increase survivalrates was probed in the colon carcinoma cell line MC38 (2×10⁶ cells)(available from Kerafast) injected subcutaneously in syngeneic C57BL/6mice shaved beforehand. The injection site was labeled with a permanentmarker. Day 2, 1×10⁷ pfu of HPV-16 E7-encoding PCPV or a MVA virus, orbuffer were injected at the cell line injection site (D2) and later inthe emerging tumor at day 9 and 16 (D9 and D16). Ten mice per group wereinjected. Tumor growth and survival were monitored over one monthfollowing MC38 cell implantation. The administration protocol isillustrated in FIG. 6A.

In all animals treated with buffer, tumors grew rapidly beyond thevolume of 1500 mm³ within the 11 to 18 days following MC38 implantation(FIG. 6D). In contrast, animals treated with the recombinantHPV16E7-encoding MVA showed a delay in tumor growth. More specifically,tumors exceeding 1500 mm³ at day 18 were observed in 5/10 whereas tumorgrowth is moderate in the 5 others (<1000 mm³ at day 18 post tumorimplantation) as illustrated in FIG. 6B. Surprisingly, PCPV treatmentprovides a stronger anti-tumoral protection as compared to MVAtreatment. Indeed, tumors reaching a volume of 1500 mm³ were observed inonly 4/10 animals after 15 to 24 days post tumor implantation and notumor growth was seen in the 6 others (FIG. 6C). On the same line, PCPVincreased survival in the treated animals (FIG. 6E)

In conclusion, when injected intratumorally into fast growing MC38tumors, PCPV injection led to tumor growth control and increasedsurvival compared to buffer or MVA injected animals.

Depletion Experiments

The tumor control experiment in MC38-bearing mice was repeated, butdepletion of specific cell populations, respectively CD4+, CD8+ andneutrophils, was applied with appropriate depleting antibodies injectedbefore and during PCPV treatment. More specifically, depletion of CD8⁺cells was carried out by intraperitoneal (ip) injection of 200 μg of arat monoclonal antibody (MAb) anti-mouse CD8 (clone 53-6-7; BioXCell) atdays −1, −2, 6 and 13. Depletion of CD4⁺ cells resulted fromintraperitoneal injection of 200 μg of a rat Mab against mouse CD4(clone GK1.5 available at BioXCell) at days −1, −2, 6 and 13. Depletionof neutrophils (and other Ly6G cells) was made according to the protocoldescribed in Wozniak et al. (2012, BMC Immunology, 13:65), using a ratMAb against mouse Ly6G (clone 1A8; BioXCell) injected ip at a dose of200 μg day −2 and then every other day or every three days over the weekend.

As described above, the colon carcinoma cell line MC38 (2×10⁶ cells) wasinjected subcutaneously in syngeneic C57BL/6 mice shaved 2 days before.The injection site was labeled with a permanent marker. One10⁷ pfu ofPCPV or buffer (negative control) were injected day 2 at the cell lineinjection site and later in the emerging tumor (days 9 and 16). 13 miceper group were injected.

Depletion of CD4⁺, CD8⁺ and Ly6G⁺ cells was confirmed in blood andspleen (periphery) from mice sacrificed at day 14 (data not shown).However, the absence of neutrophils in MC38 tumors from mice treatedwith Ly6G could not be demonstrated (Myeloperoxidase MPO signal remaineddetectable). Reduction of neutrophil counts could not be evidenced dueto the lack of quantitative measures. Depletion of CD4+ and CD8⁺ cellsin tumors could not be probed for technical reasons.

As expected, tumors grow rapidly in buffer treated animal (FIG. 12E)whereas treatment with PCPV delayed tumor growth (FIG. 12A). Tumor sizewas even more controlled between days 16 and 29 in group treated withPCPV and anti Ly6G (FIG. 12B) with respect to the group treated withPCPV alone (p-value <0.001). Treatment with PCPV and anti CD8 led tobigger tumor sizes compared to the PCPV-treated group (p-value 0.05)(FIG. 12C) whereas treatment with CD4-depleting antibody did not showsignificant effect on tumor growth (FIG. 12D). In addition, treatmentwith PCPV and anti CD8 decreased survival, while the treatment with PCPVand anti Ly6G increased survival (p-value 0.001).

The results suggest a role for CD8⁺ cells in PCPV-induced antitumoralresponse. Treatment with Ly6G increases survival, most likely due to thedepletion of circulating MDSCs and N2 neutrophils. Shaul and Fridlender(2017, J. Leuko. Biol. 102(2): 343-9), described the functionalplasticity of neutrophils (pro-tumor (N2) and anti-tumor (N1) nature ofneutrophils which infiltrate in the tumor under the influence ofspecific cytokines.

Induction of Tumor-Specific T Cells in Long Term Survivors

The presence of MC-38-specific T cells was investigated in long-termmice survivors with reduced/resolved MC38 tumors at day 34 and comparedto naive animals. Splenocytes were isolated and processed essentially asfollows. Spleens from 5 survivor animals were collected and pooled (PCPVsurvivor pool) or individual spleen of 2 survivor mice was collected inComplete medium (CM) and then crushed with a syringe plunger. Splenocytesuspension was diluted in CM, laid over 4 mL of Lympholyte®-M separationcell media (Cedarlane). The interphase containing lymphocytes wascollected by centrifugation and resuspended in RBC lysis buffer (BDPharmLyse, BD BioScience) till lysis of red blood cells (RBC) occurs.Lymphocytes were resuspended in CM and counted (Beckman Coulter) Cellconcentration was adjusted to 1×10⁷ cells per mL with CM. Stimulationwas performed with MC38 cells treated with Mitomycin C, cells stimulatedwith medium (negative control) or the irrelevant peptide i8L (negativecontrol). ELISpot analysis for antigen-specific IFN-gamma (IFNg)producing splenocytes was carried out according to standard protocol.

FIG. 13 illustrates that after PCPV treatment, tumor-specific(MC-38)-specific T cells were detected in splenocytes of pooled orindividual survivor mice (having reduced/resolved MC38 tumors at day 34)after stimulation with MC 38 cells treated with Mitocytin C. Incontrast, negative controls (stimulation with CM or irrelevant peptide)or naive mice did not show any IFNg-producing cells.

Oncolytic Potential of PCVP

The oncolytic potential of PCPV was studied in a variety of murine andhuman tumor cell lines obtained from the American Type CultureCollection (ATCC, Rockville, Md.) such as LoVo (ATCC® CCL-229™), HCT 116(ATCC® CCL-247⁷m), glioblastoma human cancer cell line U-87 MG (ATCC®HTB-14), cervix human cancer cell line HeLa (ATCC® CCL-2™) andMIA-Paca-2 (ATCC® CRL-1420™) (data not shown,) as well as the murinecolon carcinoma MC38 cell line. In all these cell lines, PCPV showed amild or no oncolytic activity Here, we show the viability of MC38 cellsinfected at MOI 1, 10⁻¹, 10⁻² and 10⁻³ with PCPV-GFP or a VV engineeredto express GFP (VVTG) as positive control. Five days later, cells wereharvested by scraping and number and proportion of living cells weredetermined using Trypan Blue-staining and the cell counter Vicell. Asshown in FIG. 7, VV infection of MC38 cells strongly impaired MC38viability (55% and 30% of cells still viable at MOI 0.1 and 1). Nineteenpercent of MC38 were infected with PCPV-GFP but the virus showed nolytic effects in the infected cells (as evidenced by the same number oflive cells at increasing from MOI 10⁻³ to 1). Mock treatment has asexpected no effect on MC38 viability.

Based on these results, one may anticipate that tumor control observedwith PCPV cannot be explained with oncolytic virtues of this virus inMC38 cells. Therefore, other correlates with in vivo efficacy of PCPVwere studied, like cytokine/chemokine profile and tumor infiltratinglymphocytes (TILs).

Local Cytokine Profile In Vivo after Subcutaneous (sc) Application ofMVAN33.1 or PCPV

For local cytokine and chemokine detection, mice were injected in bothflanks applying 5·10⁵ pfu/flank. Two skin samples per mouse were cutinto small pieces in 500 μl PBS in C-type tubes (Miltenyi Biotec), andmechanically dissociated (GentleMACS; Miltenyi Biotec). Aftercentrifugation at 300 g, supernatants were transferred in Eppendorftubes and centrifuged at 18000 g in the cold. Cleared supernatant wasanalyzed with Procartaplex mouse chemokine and cytokine multiplex kitsusing a MagPix device according to the manufacturer's recommendations.

Four mice per group were analyzed. Profiles obtained after injection ofMVA and PCPV were compared to buffer (negative control). In general,each analyte was secreted to higher extents after injection of PCPV thanafter MVA or buffer. As expected treatment with buffer had no effect onthe cytokine secretion. Surprisingly, compared to MVAN33.1, PCPV inducedsignificantly higher local levels of IP-10, IFN-gamma, TNF-alpha, IL-12,IL-1 beta, RANTES, IL-18, MCP-1 and MIP1-beta (as illustrated in FIG. 8)as well as of IL-4, GM-CSF, MIP-1alpha, Eotaxin, IL-6, GRO-alpha, MCP-3,G-CSF, M-CSF and LIF (data not shown).

Effect of PCPV on TILs in Murine MC38 Model

Mice were split into 3 groups of 6 animals. Palpable tumors grown aftersc implementation of MC38 cells (palpable tumors of at least 100 mm³)were injected intratumorally 9 days after tumor cell implantation with1×10⁷ pfu of recombinant PCPV or MVA (both viruses encode HPV16E7m, anantigen irrelevant for this tumor model) or buffer (negative control).The day after virus injection, mice were sacrificed, tumors wereisolated, enzymatically dissociated (Miltenyi products: tumordissociation kit, C tube, Gentle Macs program: Tumors m_imp tumor 02followed by 40 min at 37° C. under agitation terminated with programm_imptumor_03 twice) and TILs were enriched. After filtration ofresulting cell suspension (70 μm pores) and erythrocyte lysis (BDPharmLyse, 5 min), CD45⁺ cells were enriched using magnetic beadstechnology (Miltenyi products: CD45 TILs MicroBeads, separation onMultiMAVS Cell24, program: prog POSSEL+bloc 24). Subpopulations withinthe CD45⁺ enriched cells were identified by flow cytometry according toBrauner et al. (AACR 2017 poster abstract N^(o) 1672). The percentage ofneutrophils was detected in the population of CD11b⁺ Ly6G⁺ CD45⁺ cells.TAMs were identified as CD11c− subpopulation within Ly6G⁻CD11b⁺ cells.Various subpopulations were stratified with MHC II and Ly6C. “TAM C”according to Brauner et al, were characterized as MHC II^(lo) and Ly6C.

FIG. 9 illustrates the ability of PCPV to enhance the percentage ofneutrophils in growing tumors (9A) while decreasing the percentage ofTAM C population (9B). These results could be indicative ofPCPV-mediated tumor control.

There is also growing appreciation of neutrophil heterogeneity incancer, with distinct neutrophil populations promoting cancer control orprogression (reviewed in Singel and Segal, 2016, Immunol Rev. 273:329-43). A further characterization of subpopulations may be useful, aswell as their impact on CTL population and functions.

Concerning TAM C macrophages (MHC 11¹⁰), Movahedi et al. (2010, CancerRes70: 5728-39) describe different TAM subsets in mouse mammary tumors.MHC II^(lo) TAMS are more of a M2-like phenotype, are enriched inhypoxic tumor areas, have superior proangiogenic activity in vivo, andincreased in numbers as tumors progressed. Reducing this populationcould be indicative for a better prognostic of tumor control.

Example 4: In Vivo Immunogenicity Studies Using PCPV Expressing HPV-E7m

ELISPOT Analysis

The immunogenicity induced by PCPV vaccine vector was assessed byELISPOT and compared to MVA, the animals were divided in 4 groups of 6mice, one treated with PCPV encoding HPV16 E7mut, one with its MVAcounterpart (MVA-HPV16E7mut), one treated with empty MVA (MVA N33) and anegative group receiving buffer. 1×10⁵ pfu of virus were injectedintravenously at day 1 and day 8 into C57BL/6 mice. One week after thesecond immunization (at day 15), mice were sacrificed and spleens andlungs isolated, Spleens of all mice from one group were pooled forELISPOT analyses. Lungs were either pooled (exp ICS24) or testedindividually (exp ICS28) for antigen-specific CD8+ T cells byintracellular cytokine staining. Antigen-specificity was probed in bothtests with the HPV16E7-specific peptide R9F, the MVA-specific peptideT8V, and the irrelevant peptides I8L or K9i-3C CTRL.

ELISPOT analysis for antigen-specific IFN-gamma producing splenocyteswas carried out according to standard protocol.

Plate Preparation

Plates (Millipore, MSIPS4W10) were pre-treated 1 minute with Ethanol 35%(15p1/well) and washed five times with sterile water (200p1/well). Wellswere coated with 100p1 of 15 μg/ml anti-mouse IFNg antibody (Mabtech,AN18, 3321-3-1000; diluted in PBS) and incubated overnight or overweek-end at +4° C. The day of the experiment, plates were washed fivetimes with sterile PBS (200 μL/well) and saturated for at least 1 h at37° C. with complete medium (CM 200 μL/well).

Sample Preparation

Ex vivo Elispots were performed with fresh splenic lymphocytes. For eachexperiment, spleens from 5 animals of each group were collected andpooled. Spleens were collected in 4 mL of complete medium (CM; X-Vivo,Lonza BE04-380Q) and then crushed with a syringe plunger through a 70 μmcell strainer in a 6-well plate. Splenocyte suspension obtained wasdiluted 2-fold in CM, laid over 4 mL of Lympholyte®-M separation cellmedia (Cedarlane, ref: CL5035) and centrifuged for 20 minutes at 1500 gat room temperature. The interphase containing lymphocytes was collectedand washed twice in 10 mL of PBS (centrifugation 5 minutes at 400 g atroom temperature). Lymphocytes were resuspended in 2 mL of red bloodcell (RBC) lysis buffer (BD PharmLyse, BD BioScience, ref 555899) andincubated for 5 to 15 minutes at room temperature to lyse red bloodcells. After one wash step in CM (centrifugation 5 minutes at 400 g atroom temperature) lymphocytes were resuspended in 10 mL of CM andcounted using the «Z2 Coulter particle count and size analyzer» ofBeckman Coulter. Cell concentration was adjusted to 1×10⁷ cells per mLwith CM.

Assay

First, saturating medium was removed by emptying the plates and 50 μL CMwas added in all wells. 50 μL of each peptide solution at 4 μg/mL in CMor 50 μL of CM (negative control) were added into the relevant wells(according to a previously defined pipetting scheme; each condition wastested in quadruplicate). As positive control, 50 μL of 20 μg/mL ofConcanavalin A (ConA) were added into pre-defined wells. Secondly, 100μL of each lymphocyte suspension (1×10⁶ cells) were added into the wells(with the exception of T8V wells where only 3×10⁵ cells were added) andplates were incubated for 18 h to 20 h at 37° C. in 5% CO₂.

The following day, cells were removed by emptying the plates, plateswere washed 5 times with PBS (200 μL/well) and biotinylated anti-mouseIFNg monoclonal antibody (Mabtech, R4-6A2, 3321-6-1000; 1 μg/mL finalconcentration in PBS 0.5% FCS) was distributed in each well (100μL/well). Plates were incubated for 2 hours at room temperature, thenwashed five times with PBS and Extravidin-Phosphatase alkaline (SIGMA,E236, 1/5000e in PBS 0.5% FCS) was distributed in each well (100μL/well). Plates were again incubated for 1 hour at room temperature,washed five times with PBS and 100p1 of BCIP/NBT substrate solution(BCIP/NBT tablets, SIGMA, B5655; 0.45 μM filtered) were distributed ineach well. Plates were incubated at room temperature, in darkness, untildistinct spots were seen in positive wells (for 5 to 10 minutes). Colordevelopment was stopped by emptying the plates and extensive washings inwith tap water. Plates were left in darkness without lid until theydried for at least 1 h at room temperature.

Data Acquisition

Spots were counted with an ELISpot reader (CTL Immunoqpot reader, S5UV).A quality control was performed for each well to ensure that the countsprovided by the ELISpot reader match with the reality of the picture.Results were expressed for each quadruplicate as the mean number of spotforming units (sfu) per 1×10⁶ splenic lymphocytes. Positivity wasdetermined by the R script (livelink Biostat).

Results

Splenocytes taken from the group of animals treated withHPV16E7m-encoding PCPV and MVA showed R9F specific IFNγ secretion asillustrated in FIG. 10A, bringing to light the capacity of the tworecombinant viruses to elicit a specific immune response directed to theencoded antigen. On the other hand, no IFNγ secretion could be detectedafter administration of the empty MVA or buffer or following stimulationwith the irrelevant peptide K9i-3. On the other hand, only MVA vectors(either empty and recombinant MVA) reply following stimulation with theMVA-specific peptide T8V (FIG. 10B).

ICS Analysis on Pooled Lung Samples

The lungs of immunized mice were either pooled (ICS 024) or treatedindividually (ICS 028), as described in Remy-Ziller et al. (2018, HumVaccin Immunother. 14: 140-5). First, lungs were enzymatically andmechanically dissociated (Miltenyi products: tumor dissociation kit,C-tubes and GentleMacs). Two×10⁶ cells were stimulated ex vivo in 150 μlTexMACS medium (Miltenyi) in the presence of 1 μg anti CD28 (abcam), andeither the HPV16E7-specific peptide R9F, the MVA-specific peptide T8V,or the irrelevant peptide I8L. After five hours of incubation in thepresence of Brefeldin, cells were analysed by flow cytometry using antiCD3 and anti CD8 antibodies. After permeabilization (Cytofix/Cytoperm,BD Bioscience), activation was assessed by intracellular staining withanti-IFN-gamma-FITC (clone XMG1.2, BD Pharmingen) or its isotypecontrol. A lymphocyte subpopulation was defined as CD3^(dim)CD8^(dim)subpopulation comprising short-lived effecter cells and early effectorcells. Dot blot analysis shows that two injections of recombinant MVA orPCPV lead to the appearance of a CD3^(dim)CD8^(dim) population (asdescribed in Remy-Ziller et al, 2018, Hum Vaccin Immunother. 14: 140-5)composed of effector CD8⁺ T cells (data not shown). The extent to whichthis population was expanded was superior for MVA than PCPV FIG. 10C).

Nevertheless, comparable numbers of R9F-specific T cells were detectedin these subpopulations after injections of HPV16 E7m-encoding PCPV orMVA as shown in FIG. 10D. In other terms, the absolute number ofR9F-specific IFN-gamma-secreting cells was comparable in animalsadministered with HPV16E7m-encoding PCPV and MVA, even though theoverall number of CD3^(dim)CD8^(dim) cells induced by PCPV was lower.This suggests that the fold-induction of HPV16 E7-specific CD8+ T cellswas higher after vaccination with the PCPV vector than with the MVAvector.

Individual lungs of mice vaccinated with MVAN33.1, HPV16E7-encoding MVAand HPV16E7-encoding PCPV (i.e; non-oncogenic version thereof) wereanalyzed in terms of fold increase of R9F-specific IFN-gamma-secretingCD8+ T cells within the CD3^(dim)CD8^(dim) population. As shown in FIG.11, a significant higher (fold increase) number of counts was found inHPVE7 PCPV-treated group compared to HPVE7 MVA-treated group.

All together, these data showed that like MVA-E7, PCPV-E7 induced astrong cellular response (ELISPOT on splenocytes, and frequency ofantigen-specific short-lived effector cells), but PCPV-E7 displayed adifferent profile at the site of injection, with increased levels ofpro-immune cytokines including IP-10, IFN-gamma, GM-CSF, IL-18, MIP-1alpha, MIP-1 beta, IL-12 and IL-6. When injected intratumorally intofast growing MC-38 tumors, PCPV led to tumor control. Analysis of tumorsinfiltrates showed that PCPV treatment led to higher levels ofneutrophils and decreased frequency of MHCII^(lo) (M2) TAMs.

In conclusion, our data demonstrate that PCPV might display betterproperties than current viral vectors, in terms of local response andpriming activity, of ability to induce effector T cells and to reshapethe tumor infiltration profiles. Although many gene differences comparedto other poxviruses, PCPV has the capacity to encode and deliver largegenetic payload, which be useful for designing advanced anti-tumorvaccines.

Heterologous Prime Boost (PCPV/MVA)

It was published that patients with PCPV infection do not developimmunity to vaccinia/MVA and vice versa (Friedman-Kien et al., 1963,Science 140: 1335-6). Combination treatment with MVA-HPV16E7 andPCPV-HPV16E7 was thus studied in TC1 tumor model.

To this, HPV16E7-positive TC1 cells were subcutaneously implanted in theflank of C57BL/6 mice. After 14 days, tumor-bearing mice were randomized(10 mice per group), and intratumorally injected with 1×10⁶ pfu ofMVA-HPV16E7 or PCPV-HPV16E7. One week later, boost was carried out byintravenous injection with 1×10⁶ pfu of PCPV-HPV16E7 or MVA-HPV16E7.Tumor growth and survival were followed over time in mice treated withhomologous MVA/MVA and heterologous MVA/PCPV and PCPV/MVA settings orwith buffer under the same conditions (intratumoral prime and iv boost)as negative control.

As illustrated in FIG. 14, heterologous treatments resulted in bettertumor control compared to treatment with homologous MVA-HPV16E7 primeboost. Interestingly, both MVA prime/PCPV boost or PCPV prime/MVA boostproduced a decline in tumor growth with respect to MVA prime/MVA boostgroup although PCPV prime/MVA boost trends to better control tumorgrowth (tumor control in a higher number of mice as shown in FIG. 14C).As expected, tumor grew quite rapidly in buffer treated animals.

Example 5: Combo Treatment of PCPV with an ICI (Immune CheckpointInhibitor)

The effect of combining PCPV treatment with systemic ICI treatment wasevaluated in the MC38-tumor model. The rat anti mPD-1 (m for mouse)antibody RMP1-14 (commercially available from BioXcell) was chosen. Thisantibody was shown to block the interaction of mPD1 with its ligands(Yamazaki et al., 2005, J. Immunol. 175(3): 1586-92).

On day 1 (D1), the colon carcinoma cell line MC38 (2×10⁶ cells) wasinjected subcutaneously (sc) in the right flank of syngeneic C57BL/6mice shaved 2 days before. The injection site was labeled with apermanent marker. The same day, four-time less MC38 cells (5×10⁵) wereinjected sc in the left flank. One 10⁷ pfu of PCPV (PCPTG19178) or VVvirus (VVTG5095), both encoding HPV16E7m (irrelevant for the MC38model), or buffer (S08) were injected intratumorally (it) at the cellline injection site on the right side (day 2) and later in the emergingright tumors (days 9 and 16). On the other hand, 200 μg of anti-mPD-1(RMP1-14) was injected intra peritoneally (ip) at days 5, 9 and 16.Tumor growth of right (injected) and left tumors and survival werefollowed over time.

Altogether, the animals were divided in 6 groups of 13 mice,respectively:

-   -   a control group receiving S08 buffer (3 it injections at days 2,        9 and 16),    -   a group of mice treated with the E7-expressing PCPV virus (3 it        injections of 10⁷ pfu of PCPTG19178 at days 2, 9 and 16),    -   a group of mice treated with the E7-expressing oncolytic VV        virus (3 it injections of 10⁷ pfu of VVTG5095 at days 2, 9 and        16),    -   a group of mice treated with the anti PD1 antibody (3 ip        injections of RMP1-14 antibody at days 5, 9 and 16,    -   a group receiving both the E7-expressing PCPV virus and the anti        PD1 antibody (3 it injections of 10⁷ pfu of PCPTG19178 at days        2, 9 and 16 and 3 ip injections of RMP1-14 antibody at days 5, 9        and 16), and    -   a group receiving both the E7-expressing VV oncolytic virus and        the anti PD1 antibody (3 it injections of 10⁷ pfu of VVTG5095 at        days 2, 9 and 16 and 3 ip injections of RMP1-14 antibody at days        5, 9 and 16).

FIG. 15 shows diameter evolution of the tumors implanted on the rightflanks in the groups of mice treated with PCPV (A) or VV (B) either asstand alone or in combination with the murine anti-PD1. Morespecifically, tumors grew rapidly in the control group (S08) goingbeyond 2000 mm³ on average as soon as D13 whereas treatment withanti-mPD1 allowed to postpone the average 2000 mm³ volume of some days(D16). As illustrated in FIG. 15B, the same tendency was observedfollowing treatment with the oncolytic VVTG5095 (dark grey). But tumorgrowth was controlled in mice treated with VVTG5095 and anti-mPD1 incombination (middle grey) where average 2000 mm³ volume was obtained atD22. In contrast, as shown in FIG. 15A, tumor growth is significantlydelayed following treatment with PCPV virus (dark grey) where theaverage tumor volume was well below 2000 mm³ at D22 and even more in thecombo group (medium grey) receiving both PCPTG19178 and anti-PD1.Notably, in combo PCPV-HPV16E7+anti PD-1 group, difference in tumor sizewas found significant versus the tumor sizes obtained in the three othergroups PCPV-HPV16E7, anti PD-1 and S08.

In contralateral tumors (left flank), treatment with the anti-mPD1antibody delayed tumor progression. A significant tumor size reductionwas found in anti-PD1 group versus the other groups (data not shown).

For Survival analysis (data not shown), a significant higher OS wasfound in both PCPV-HPV16E7 group and PCPV-HPV16E7+anti PD-1 combo groupversus S08 buffer group (adjusted p-values were both 0.036). Meansurvival differences were respectively 4.4 days and 3.4 days. Asignificant higher OS was also found in VV-HPV16E7+anti PD-1 combo groupversus VV-HPV16E7 and control groups (adjusted p-values were both 0.040and 0.004). Mean survival differences were respectively 2.9 days and 3.6days.

These results highlight that combining a recombinant PCPV with an ICIsuch as anti-PD1 allows to strengthen the anti-tumor protection.

Example 6: Effect of Bovine Popular Stomatitis Virus

The Parapoxvirus genus comprises distinct members including the ORFvirus, PCPV and the bovine papular stomatitis virus (BPSV), all cancause infections of ruminants and their handlers (Zhao et al, 2013, JVirol Methods, 194:229-34). PAPV infection in humans induces vigorousand short-lived cell-mediated immune response and a poor and short-livedhumoral response, about 8-12% of individuals have second infectionshandlers (Zhao et al., 2013, J Virol Methods, 194:229-34).

Another member of Parapoxvirus genus, (BPSV) was tested for its abilityto induce the secretion of IFN alpha in PBMCs obtained from 2 differentdonors and compared to MVA and PCPV (see Example 1). Fresh PBMCs wereisolated, incubated overnight (resting) and infected next day with thevirus at MOI 0.3. A CpG type TLR9 ligand (ODN2216 obtained fromInvivogen) was added as a control for pDC, B cell-mediated secretion ofIFN-alpha. Supernatants were taken 2, 4, 6, 16 or 24 h after infection,IFN-alpha in the supernatant was quantified by Luminex technology asdescribed above.

As shown in FIG. 16, BPSV-infected PBMCs as PCPV-infected PBMCs inducedthe secretion of very high levels of IFN-alpha at 16 and 24 h post cellinfection. The secreted IFN-alpha levels in both cases were well abovethe moderate secretion levels of IFN-alpha observed upon MVA infectionconfirming the results obtained in Example 1 and above the ODN2216control.

In conclusion, BPSV showed similar effects as PCPV, i.e. strong increaseof secreted IFN-alpha in supernatant of PBMCs obtained from variousdonors.

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1. A pseudocowpoxvirus (PCPV) wherein said PCPV comprises at least oneforeign nucleic acid inserted in its genome.
 2. The PCPV of claim 1,wherein said PCPV is obtained from the wild-type TJS strain asidentified by ATCC reference number ATCC VR-634™ or from a virus strainof the same or similar name or functional fragments and variantsthereof.
 3. The PCPV of claim 1, wherein said PCPV is further defectivefor a viral function encoded by the PCPV genome.
 4. The PCPV of claim 1,wherein said foreign nucleic acid encodes a polypeptide selected fromthe group consisting of polypeptides that compensate for defective ordeficient proteins in a subject, suicide gene products, armed geneproducts, immunostimulatory polypeptides, and antigenic polypeptides. 5.The PCPV of claim 4, wherein said immunostimulatory polypeptide isselected from the group consisting of cytokines, chemokines,interferons, tumor necrosis factor, colony-stimulating factors,APC-exposed proteins, agonists of immune checkpoints, and antagonists ofimmune checkpoints.
 6. The PCPV of claim 5, wherein saidimmunostimulatory polypeptide is GM-CSF or is an agonist OX40-directedantibody.
 7. The PCPV of claim 4, wherein said antigenic polypeptide isa cancer antigen, or is a viral antigenic polypeptide.
 8. (canceled) 9.The PCPV of claim 4, wherein the at least one foreign nucleic acid isplaced under the control of a vaccinia virus promoter one selected fromthe group consisting of the 7.5K, H5R, 11K7.5, SE, TK, pB2R, p28, p11,and K1L promoter, synthetic promoters, and early/late chimericpromoters.
 10. The PCPV of claim 1, wherein the at least one foreignnucleic acid is inserted in the VEGF locus.
 11. A method for generatingthe PCPV of claim 1, by homologous recombination between a transferplasmid comprising the foreign nucleic acid flanked in 5′ and 3′ withPCPV sequences respectively present upstream and downstream theinsertion site and a PCPV genome, wherein said method comprises a stepof generating said transfer plasmid and a step of introducing saidtransfer plasmid into a suitable host cell. 12.-19. (canceled)
 20. Amethod for amplifying the PCPV according to claim 1 or generated by amethod for generating a PCPV as defined in claim 11, comprising thesteps of a) preparing a producer cell line, b) transfecting or infectingthe prepared producer cell line, c) culturing the transfected orinfected producer cell line under suitable conditions so as to allow theproduction of the virus, d) recovering the produced virus from theculture of said producer cell line and optionally e) purifying saidrecovered virus.
 21. (canceled)
 22. A composition comprising atherapeutically effective amount of the PCPV according to claim 1 and apharmaceutically acceptable vehicle.
 23. The composition of claim 22,wherein said composition is formulated in individual doses comprisingfrom approximately 10³ to approximately 10¹² pfu of PCPV.
 24. Thecomposition of claim 22, wherein the composition is formulated forintravenous, intramuscular, subcutaneous, or intratumoraladministration.
 25. (canceled)
 26. A method of treatment comprisingadministering the composition of claim 22 to a subject in need thereofin an amount sufficient to treat or prevent a disease or a pathologicalcondition caused by a pathogenic organism or an unwanted cell division.27. The method according to claim 26 wherein said method of treatment ofa disease or a pathological condition caused by an unwanted celldivision is a method for inhibiting tumor cell growth.
 28. The methodaccording to claim 26, comprising 2 to 6 weekly administrations possiblyfollowed by 2 to 15 administrations at 3 weeks interval of the PCPVcomposition comprising 10⁶ to 10⁹ pfu. 29.-34. (canceled)
 35. A methodfor eliciting or stimulating and/or re-orienting an immune responsecomprising administering the composition according to claim 22 to asubject in need thereof, in an amount sufficient to activate thesubject's immunity.
 36. The method according to claim 35, resulting inat least one the following properties: The secretion of high levels ofIFN-alpha from PBMC; The activation of monocyte-derived dendritic cells;The induction of T cell activation or proliferation; A bettercytokine/chemokine profile in MDSC; activation of APC; a M2 to M1conversion of human macrophages; and/or Induction of immunity through aTLR9-mediated pathway or others innate immunity-stimulating pathways.37. The method according to claim 27, comprising 2 to 6 weeklyadministrations possibly followed by 2 to 15 administrations at 3 weeksinterval of the PCPV composition comprising 10⁶ to 10⁹ pfu.
 38. Themethod according to claim 37, wherein said method is for treating acancer selected from the group consisting of renal cancer, prostatecancer, breast cancer, colorectal cancer, lung cancer, liver cancer,gastric cancer, bile duct carcinoma, endometrial cancer, pancreaticcancer, ovarian cancer, and a cancer that overexpresses MUC1.
 39. Themethod according to claim 38, for treating a subject having a non-smallcell lung cancer (NSCL) using a PCPV encoding the MUC1 antigen.
 40. Themethod according to claim 38, for treating a HPV-positive cancer using aPCPV encoding a HPV16 E7 antigen.
 41. The method according to claim 37,which is used in conjunction with one or more other therapeutic agentsselected from the group consisting of surgery, radiotherapy,chemotherapy, cryotherapy, hormonal therapy, toxin therapy,immunotherapy, cytokine therapy, targeted cancer therapy, gene therapy,photodynamic therapy, and transplantation.
 42. The method according toclaim 41, which is carried out according to a prime boost approach whichcomprises sequential administrations of a priming composition(s) and aboosting composition(s).
 43. The method according to claim 42, whereasthe priming composition is a PCPV composition administered byintratumoral route and the boosting composition is a MVA compositionadministered by intravenous route.
 44. The PCPV of claim 7, wherein saidcancer antigen is a MUC-1 antigen, or wherein said viral antigenicpolypeptide is a HPV-16 E7 antigen or HBV antigen.